Targeted therapeutics based on engineered proteins that bind egfr

ABSTRACT

The present invention relates to single domain proteins that bind to epidermal growth factor receptor (EGFR). The invention also relates to single domain proteins for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such proteins, polynucleotide encoding such proteins or fragments thereof, and to vectors comprising the polynucleotides encoding the innovative proteins.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/065,955, filed Feb. 14, 2008. All the teachings of theabove-referenced application are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to single domain proteins that bind toepidermal growth factor receptor (EGFR). The invention also relates tosingle domain proteins for use in diagnostic, research and therapeuticapplications. The invention further relates to cells comprising suchproteins, polynucleotide encoding such proteins or fragments thereof,and to vectors comprising the polynucleotides encoding the innovativeproteins.

INTRODUCTION

The HER family of receptor tyrosine kinases are important mediators ofcell growth, differentiation and survival. The receptor family includesfour distinct members including epidermal growth factor receptor (EGFR,ErbB1, or HER1), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).

The HER family has been causally implicated in human malignancy.Abnormal activity of the Her family of receptors is involved with breastcancer. EGFR, Her-3, and Her-4 are frequently expressed in ovariangranulosa cell tumors (Leibl, S. et al., Gynecol Oncol 101:18-23 (2005).In particular, increased expression of EGFR has been observed in breast,bladder, lung, head, neck and stomach cancer as well as glioblastomas.

Increased EGFR receptor expression may be associated with increasedproduction of a EGFR ligand, transforming growth factor alpha (TGF-α),by the same tumor cells resulting in receptor activation by an autocrinestimulatory pathway. Baselga and Mendelsohn Pharmac. Ther. 64:127-154(1994). Monoclonal antibodies directed against the EGFR or its ligandshave been evaluated as therapeutic agents in the treatment of suchmalignancies. See, e.g., Baselga and Mendelsohn., supra; Masui et al.Cancer Research 44:1002-1007 (1984); and Wu et al. J. Clin. Invest.95:1897-1905 (1995).

HER receptors generally reside in various combinations in cells. Theirheterodimerization is thought to increase the diversity of cellularresponses to a variety of HER ligands (Earp et al. Breast CancerResearch and Treatment 35: 115-132 (1995)). EGFR is bound by at leastsix different ligands; epidermal growth factor (EGF), transforminggrowth factor alpha (TGF-α), amphiregulin, heparin binding epidermalgrowth factor (HB-EGF), betacellulin and epiregulin (Groenen et al.Growth Factors 11:235-257 (1994)).

EGF binds to EGFR, which forms a heterodimer with HER2, activating EGFRand resulting in transphosphorylation of HER. Dimerization and/ortransphosphorylation activates the HER2 tyrosine kinase.

The potential side effects of therapeutics is an important issue toconsider when devising a treatment regiment. As an example, cetuximab(Erbitux™), an anti-EGFR antibody, has been associated with potentiallylife threatening infusion reactions (Thomas, M., Clin J Oncol Nurs.9(3):332-8 (2005)). Gefitinib (Iressa™) and erlotinib (Tarceva™), bothEGFR specific small molecule inhibitors, are associated with a risk ofinterstitial lung disease (Sandler A., Oncology 20(5 Suppl 2):35-40(2006)). Individual patients may be predisposed to particular types ofcomplications that affect the choice of drug treatment. Offering agreater choice of treatment options allows physicians to select thetherapeutic with the best side effect profile for an individual patient.The present invention provides novel polypeptides and proteintherapeutics useful in methods of treatment.

In view of the role that EGFR signaling plays in disorders, includingcancer and proliferative disorders, it would be desirable to generatetherapeutics, such as EGFR binding polypeptides, that selectivelymodulate, inhibit or block EGFR.

In addition, it would be desirable for such a therapeutic to beexpressed in a cost effective manner, possess desirable biophysicalproperties (e.g. Tm, substantially monomeric, or well folded), have asmall size to penetrate tissues, and have a suitable half life in vivo.

SUMMARY OF THE INVENTION

One aspect of the application provides a polypeptide comprising afibronectin type III (Fn3) domain, wherein the Fn3 domain (i) comprisesa loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop EF; and a loopFG; (ii) has at least one loop selected from loop BC, DE, and FG with analtered amino acid sequence relative to the sequence of thecorresponding loop of the human Fn3 domain, and (iii) binds humanepidermal growth factor receptor (EGFR). In some embodiments, thepolypeptide binds EGFR with a K_(D) of less than 10⁻⁴M, 10⁻⁵M, 10⁻⁶M,10⁻⁷M, 10⁻⁸M, 10⁻⁹M, or 10⁻⁹M. In some embodiments, at least two loopsof the Fn3 domain are altered. In some embodiments, loop BC and loop FGhave an altered amino acid sequence relative to the sequence of thecorresponding loop of the human Fn3 domain. In some embodiments, atleast three loops of the Fn3 domain are altered. In some embodiments, atleast two loops of the Fn3 domain bind EGFR. In some embodiments, atleast three loops of the Fn3 domain bind EGFR. In some embodiments, atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in at least one loopselected from loop BC, DE, and FG are substituted with an amino acidthat differs from the wild-type sequence. In some embodiments, at least1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted or added to atleast one loop selected from loop BC, DE, and FG. In some embodiments,the EGFR binding polypeptide binds to a related receptor, such as HER2or HER3, with a K_(D) of more than 10⁻⁶M, 10⁻⁵M, 10⁻⁴M, 10⁻³M, or 10⁻²M.In some embodiments, the EGFR binding polypeptide inhibits EGFR bindingto one or more EGFR ligands. In some embodiments, the EGFR bindingpolypeptide inhibits EGFR signaling.

In some embodiments, the EGFR binding polypeptide is a tenth fibronectintype III domain (¹⁰Fn3). In some embodiments, the ¹⁰Fn3 comprises theamino acid sequence of any one of SEQ ID NOS: 207-231. In someembodiments, the ¹⁰Fn3 comprises the amino acid sequence of SEQ ID NO:215. In some embodiments, the ¹⁰Fn3 comprises the amino acid sequence atleast 75, 80, 85, 90, 95, or 98% identical to any one of SEQ ID NOS:207-231.

In some embodiments, the EGFR binding polypeptide further comprises oneor more pharmacokinetic (PK) moieties selected from: a polyoxyalkylenemoiety, a human serum albumin binding protein, sialic acid, human serumalbumin, transferrin, IgG, an IgG binding protein, and an Fc fragment.In some embodiments, the PK moiety is the polyoxyalkylene moiety andsaid polyoxyalkylene moiety is polyethylene glycol (PEG). In someembodiments, the PEG moiety is covalently linked to the EGFR bindingpolypeptide via a Cys or Lys amino acid. In some embodiments, the EGFRbinding polypeptide is a Fn3 domain. In some embodiments, the PEG isbetween about 0.5 kDa and about 100 kDa.

In some embodiments, the PK moiety improves one or more pharmacokineticproperties of the polypeptides, e.g., bioavailability, serum half-life,in vivo stability, and drug distribution. In some embodiments, the PKmoiety increases the serum half-life of the EGFR binding polypeptide byat least 20, 30, 40, 50, 70, 90, 100, 120, 150, 200, 400, 600, 800% ormore relative to the EGFR binding polypeptide alone. In someembodiments, the EGFR binding polypeptide further comprising a PK moietyhas a serum in vivo half-life of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, or 14 days.

In some embodiments, the PK moiety and the Fn3 domain are operablylinked via at least one disulfide bond, a peptide bond, a polypeptide, apolymeric sugar, or a polyethylene glycol moiety. In some embodiments,the PK moiety and the Fn3 domain are operably linked via a polypeptidecomprising the amino acid sequence of SEQ ID NOS: 232-235.

In some embodiments, the EGFR binding polypeptide further comprises asecond domain. In some embodiments, the EGFR binding polypeptide furthercomprises an antibody moiety. In some embodiments, the antibody moietyis less than 50 KDa. In some embodiments, the antibody moiety is lessthan 40 KDa. In some embodiments, the antibody moiety is a single chainFvs (scFvs), Fab fragment, Fab′ fragment, F(ab′)₂, disulfide linked Fv(sdFv), Fv, diabody, or whole antibody. In some embodiments, theantibody moiety is a single domain antibody. In some embodiments, theantibody moiety binds a human protein. In some embodiments the antibodymoiety binds IGF-IR, FGFR1, FGFR2, FGFR3, FGFR4, c-Kit, human p185receptor-like tyrosine kinase, HER2, HER3, c-Met, folate receptor,PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascular endothelial growth factor(VEGF) A, VEGF C, VEGF D, human CD20, human CD18, human CD11a, humanapoptosis receptor-2 (Apo-2), human alpha4beta7 integrin, humanGPIIb-IIIa integrin, stem cell factor (SCF), EGFR, or human CD3.

In some embodiments, the EGFR binding polypeptide further comprises aderivative of lipocalin; a derivative of tetranectin; an avimer; or aderivative of ankyrin. In some embodiments, the EGFR binding polypeptidebinds a human protein. In some embodiments the EGFR binding polypeptidebinds IGF-IR, FGFR1, FGFR2, FGFR3, FGFR4, c-Kit, human p185receptor-like tyrosine kinase, HER2, HER3, c-Met, folate receptor,PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascular endothelial growth factor(VEGF) A, VEGF C, VEGF D, human CD20, human CD18, human CD11a, humanapoptosis receptor-2 (Apo-2), human alpha4beta7 integrin, humanGPIIb-IIIa integrin, stem cell factor (SCF), EGFR, or human CD3.

In some embodiments, the EGFR binding polypeptide is a Fn3 domain andfurther comprises a second Fn3 domain. The second Fn3 domain (i)comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; and a loop FG;(ii) has at least one loop selected from loop BC, DE, and FG with analtered amino acid sequence relative to the sequence of thecorresponding loop of the human Fn3 domain, and (iii) binds a humanprotein that is not bound by the human Fn3 domain. In some embodiments,the second Fn3 domain binds IGF-IR, FGFR1, FGFR2, FGFR3, FGFR4, c-Kit,human p185 receptor-like tyrosine kinase, HER2, HER3, c-Met, folatereceptor, PDGFR, VEGFR1, VEGFR2, VEGFR3, human vascular endothelialgrowth factor (VEGF) A, VEGF C, VEGF D, human CD20, human CD18, humanCD11a, human apoptosis receptor-2 (Apo-2), human alpha4beta7 integrin,human integrin, stem cell factor (SCF), EGFR, or human CD3. In someembodiments, the second Fn3 domain binds a human protein with a K_(D) ofless than 10⁻⁴M, 10⁻⁵M, 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, or 10⁻⁹M. In someembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in atleast one loop selected from loop BC, DE, and FG of the second Fn3domain are substituted with an amino acid that differs from thewild-type sequence. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 amino acids are deleted or added to at least one loopselected from loop BC, DE, and FG.

In some embodiments, the EGFR binding polypeptide is a ¹⁰Fn3 domain thatbinds EGFR further comprising a second ¹⁰Fn3 domain. In someembodiments, the second ¹⁰Fn3 domain binds IGF-IR. In some embodiments,the second ¹⁰Fn3 domain binds VEGFR2. In some embodiments, the second¹⁰Fn3 domain binds EGFR. In some embodiments, the second ¹⁰Fn3 domaincomprises the amino acid sequence of any of one of SEQ ID NOS: 2-125,184-204, or 236. In some embodiments, the second ¹⁰Fn3 domain comprisesthe amino acid sequence at least 75, 80, 85, 90, 55, or 98% identical toany of one of SEQ ID NOS: 2-125, 184-204, or 236. In some embodiments,the second ¹⁰Fn3 domain comprises the amino acid sequence of any of oneof SEQ ID NOS: 126-183, 205, or 206. In some embodiments, the second¹⁰Fn3 domain comprises the amino acid sequence at least 75, 80, 85, 90,55, or 98% identical to any of one of SEQ ID NOS: 126-183, 205, or 206.In some embodiments, the second ¹⁰Fn3 domain comprises the amino acidsequence of any of one of SEQ ID NOS: 207-231. In some embodiments, thesecond ¹⁰Fn3 domain comprises the amino acid sequence at least 75, 80,85, 90, 55, or 98% identical to any of one of SEQ ID NOS: 207-231.

In some embodiments, the EGFR binding polypeptide further comprises asecond domain operably linked via at least one disulfide bond, a peptidebond, a polypeptide, a polymeric sugar, or a polyethylene glycol moiety(PEG). In some embodiments, the PEG is between about 0.5 kDa and about100 kDa. In some embodiments, the PEG is conjugated to the polypeptideand the second domain via a Cys or Lys residue. In some embodiments, atleast the EGFR binding polypeptide or the second domain has no more thana single Cys or Lys. In some embodiments, the single Cys or Lys islocated in a non-wildtype location in the amino acid sequence. In someembodiments, the EGFR binding polypeptide comprises SEQ ID NO: 235.

In one aspect, the application provides an EGFR binding polypeptide thatinhibits the binding of transforming growth factor alpha (TGF-alpha) orepidermal growth factor (EGF) to EGFR and does not activate human EGFRat sub IC₅₀ concentrations in a cell-based assay. In some embodiments,the EGFR binding polypeptide inhibits EGFR binding to one or more EGFRligands.

In one aspect, the application provides an EGFR binding polypeptide thatpolypeptide competes with an anti-EGFR antibody for binding to EGFR. Insome embodiments, the anti-EGFR antibody is selected from panitumumab,nimotuzumab, zalutumumab, EMD72000, and cetuximab.

In one aspect, the application provides an EGFR binding polypeptide thatinhibits total EGF-stimulated phosphotyrosine activation of EGFR with anIC₅₀ of less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, or 10⁻⁹ M.

In one aspect, the application provides an EGFR binding polypeptide thatinhibits ERK phosphorylation with an IC₅₀ of less than 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, or 10⁻⁹ M.

In one aspect, the application provides an EGFR binding polypeptide thatinhibits AKT phosphorylation with an IC₅₀ of less than 10⁻⁵, 10⁻⁶, 10⁻⁷,10⁻⁸, or 10⁻⁹ M.

In one aspect, the application provides an EGFR binding polypeptide thatinduces apoptosis in a cell based assay in a cell line dependent on EGFRactivation. In some embodiments, EGFR binders block EGFR activities suchas control of apoptosis, phosphorylation or dimerization.

In one aspect, the application provides an EGFR binding polypeptide thathas been deimmunized to remove one or more T-cell epitopes. In oneaspect, the application provides an EGFR binding polypeptide that hasbeen deimmunized to remove one or more B-cell epitopes.

In one aspect, the application provides an EGFR binding Fn3 domainselected by the method comprising a) producing a population of candidatenucleic acid molecules, each comprising a candidate fibronectin type III(Fn3) domain sequence which differs from human Fn3 domain codingsequence, said nucleic acid molecules each comprising a translationinitiation sequence and a start codon operably linked to said candidateFn3 domain coding sequence and each being operably linked to a nucleicacid-puromycin linker at the 3′ end; b) in vitro translating saidcandidate Fn3 domain coding sequences to produce a population ofcandidate nucleic acid-Fn3 fusions; c) contacting said population ofcandidate nucleic acid-Fn3 fusions with EGFR; and d) selecting a nucleicacid-Fn3 fusion, the protein portion of which has a binding affinity orspecificity for EGFR that is altered relative to the binding affinity orspecificity of said human Fn3 for EGFR. In some embodiments, theselected nucleic acid-Fn3 fusion is further optimized by altering one ormore nucleic acid residues and rescreening the fusion with EGFR toselect for improved binders. In some embodiments the candidate nucleicacid molecule is RNA. In some embodiments the candidate nucleic acidmolecule is DNA. In some embodiments, the nucleic acid-puromycin isDNA-puromycin. In some embodiments, the Fn3 domain is ¹⁰Fn3.

In one aspect, the application provides pharmaceutically acceptablecompositions comprising an EGFR binding polypeptide. In someembodiments, the composition is essentially endotoxin free. In someembodiments, the compositions is substantially free of microbialcontamination making it suitable for in vivo administration. Thecomposition may be formulated, for example, for IV, IP or subcutaneousadministration. In some embodiments, the EGFR binding polypeptideinhibits EGFR binding to one or more EGFR ligands. In some embodiments,the EGFR binding polypeptide inhibits EGFR signaling.

One aspect of the application provides methods for the treatment of asubject having a cancer by administering an EGFR binding polypeptide,either alone or in combination with other cytotoxic or therapeuticagents. The cancer can be one or more of, for example, breast cancer,colon cancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostatecancer, lung cancer, synovial carcinoma, glioblastoma, pancreaticcancer, or other cancer yet to be determined in which EGFR levels areelevated, up-regulated, mutated or altered in physiology compared tonon-oncogenic cells.

One aspect of the application provides methods for the treatment of asubject having a cancer by administering an EGFR binding polypeptide,either alone or in combination with other cytotoxic or therapeuticagents. In particular, preferred cytotoxic and therapeutic agentsinclude docetaxel, paclitaxel, doxorubicin, epirubicin,cyclophosphamide, trastuzumab, capecitabine, tamoxifen, toremifene,letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin,carboplatin, cisplatin, dexamethasone, antide, bevacizumab,5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide,topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone,abarelix, zoledronate, streptozocin, rituximab, idarubicin, busulfan,chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab,tositumomab, interferon alpha-2b, melphalam, bortezomib, altretamine,asparaginase, gefitinib, erlonitib, anti-EGF receptor antibody (e.g.,cetuximab or panitumumab), ixabepilone, and an epothilone or derivativethereof. More preferably, the therapeutic agent is a platinum agent(such as carboplatin, oxaliplatin, cisplatin), a taxane (such aspaclitaxel, docetaxel), gemcitabine, or camptothecin.

Another aspect of the application provides kits comprising one or moreof the elements described herein, and instructions for the use of thoseelements. In some embodiments, a kit includes an EGFR bindingpolypeptide, alone or with a second therapeutic agent. The instructionsfor inhibiting the growth of a cancer cell using an EGFR bindingpolypeptide, alone or with a second therapeutic agent, and/orinstructions for a method of treating a patient having a cancer usingthe same.

A further aspect of the application provides for a cell, comprising apolynucleotide encoding an EGFR binding polypeptide. Vectors containingpolynucleotides for such proteins are included as well. Sequences arepreferably optimized to maximize expression in the cell type used.Preferably, expression is in E. coli. EGFR binding polypeptides can alsobe expressed, for example, in eukaryotic microbes, including yeast(e.g., pichia or cervaisea) or blue green algae. Yeast cells can beengineered to produce desired glycosylations. The cells of the inventioncan be a mammalian cell. In one aspect, the mammalian cell can beengineered to produce desired glycosylations. In one aspect, the cellexpresses a fibronectin based scaffold protein. In one aspect, thepolynucleotides encoding fibronectin based scaffold proteins are codonoptimized for expression in the selected cell type.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Selection Profile for Isolation of EGFR-Binding Clones Thepercentage of libraries from Example 1 bound to EGFR-Fc is shown aftereach selection round. Each RNA-protein fusion library bound to thetarget after 5 cycles of selection.

FIG. 2. Cell-Based Competitive Ligand Binding Assay LA-1 (anti-EGFRmonoclonal antibody) and 679F09 (EGFR binding clone) Midscale proteinpreparations diluted in either PBS buffer or Tris buffer were assayed inthe Cell-Based Competitive Ligand Binding Assay (see Material andMethods section for details). LA-1 (circles), 679F09 Midscale PBS(squares) and 679F09 Midscale Tris (triangles) all competed with Eu-EGFfor binding to EGFR on A431 cells. The IC₅₀'s were: LA-1 (2.254 nM),679F09 Miscale PBS (13.018 nM) and 679F09 Midscale Tris (20.002 nM).

FIG. 3. Cell-Based Competitive Ligand Binding Assay LA-1 (anti-EGFRmonoclonal antibody), 679F09 (EGFR binding clone, Midscale preparation)and 867A01 (EGFR binding clone, HTPP preparation) were assayed in theCell-Based Competitive Ligand Binding Assay. LA-1 (circles), 679F09(squares) and 867A01 (triangles) all competed with Eu-EGF for binding tothe EGFR on A431 cells. The IC₅₀'s were: LA-1 (6.641 nM), 679F09 (14.726nM) and 867A01 (258.258 nM).

FIG. 4. Cell-Based Competitive Ligand Binding Assay LA-1 (anti-EGFRmonoclonal antibody) and 679F03 (EGFR binding clone, Midscalepreparation) were assayed in the Cell-Based Competitive Ligand BindingAssay. LA-1 (squares) and 679F03 (triangles) competed with Eu-EGF forbinding to the EGFR on A431 cells. The IC₅₀'s were: LA-1 (6.944 nM) and679F03 (26.847 nM).

FIG. 5. Optimization of an EGFR-specific Clones Schematic of process forfurther optimization of EGFR binding clone as described in Example 4.

FIG. 6 depicts the sequences described throughout the application.

DETAILED DESCRIPTION OF THE INVENTION Definitions

By a “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.“Polypeptide,” “peptide,” and “protein” are used interchangeably herein.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

The term “single domain polypeptide” is used to indicate that the targetbinding activity (e.g., EGFR binding activity) of the subjectpolypeptide is situated within a single structural domain, asdifferentiated from, for example, antibodies and single chainantibodies, where antigen binding activity is generally contributed byboth a heavy chain variable domain and a light chain variable domain. Asingle domain polypeptide may be attached (e.g., as a fusion protein) toany number of other polypeptides, such as fluorescent polypeptides,targeting polypeptides and polypeptides having a distinct therapeuticeffect.

The term “PK” is an acronym for “pharmokinetic” and encompassesproperties of a compound including, by way of example, absorption,distribution, metabolism, and elimination by a subject. A “PK modulationprotein” or “PK moiety” refers to any protein, peptide, or moiety thataffects the pharmokinetic properties of a biologically active moleculewhen fused to or administered together with the biologically activemolecule. Examples of a PK modulation protein or PK moiety include PEG,human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos.20050287153 and 20070003549), human serum albumin, Fc or Fc fragments,and sugars (e.g., sialic acid).

A “functional Fc region” possesses at least one “effector function” of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc.Such effector functions generally require the Fc region to be combinedwith a binding domain (e.g., an antibody variable domain) and can beassessed using various assays known in the art for evaluating suchantibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature.

A “variant Fc region” comprises an amino acid sequence which differsfrom that of a native sequence Fc region by virtue of at least one aminoacid modification. Preferably, the variant Fc region has at least oneamino acid substitution compared to a native sequence Fc region or tothe Fc region of a parent polypeptide, e.g., from about one to about tenamino acid substitutions, and preferably from about one to about fiveamino acid substitutions in a native sequence Fc region or in the Fcregion of the parent polypeptide. The variant Fc region herein willpreferably possess at least about 80% sequence identity with a nativesequence Fc region and/or with an Fc region of a parent polypeptide, andmost preferably at least about 90% sequence identity therewith, morepreferably at least about 95% sequence identity therewith.

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. To assess ADCC activity of a molecule of interest, an in vitroADCC assay, such as that described in U.S. Pat. No. 5,500,362 or5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in a animal modelsuch as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc. has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

An “isolated” polypeptide is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the polypeptide willbe purified (1) to greater than 95% by weight of polypeptide asdetermined by the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, preferably, silverstain. Isolated polypeptide includes the polypeptide in situ withinrecombinant cells since at least one component of the polypeptide'snatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

Targets may also be fragments of said targets. Thus a target is also afragment of said target, capable of eliciting an immune response. Atarget is also a fragment of said target, capable of binding to a singledomain antibody raised against the full length target.

A fragment as used herein refers to less than 100% of the sequence(e.g., 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% etc.), butcomprising 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25 or more amino acids. A fragment is of sufficient lengthsuch that the interaction of interest is maintained with affinity of1×10⁻⁶M or better.

A fragment as used herein also refers to optional insertions, deletionsand substitutions of one or more amino acids which do not substantiallyalter the ability of the target to bind to a single domain antibodyraised against the wild-type target. The number of amino acid insertionsdeletions or substitutions is preferably up to 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69 or 70 amino acids.

A protein of the invention that “induces cell death” is one which causesa viable cell to become nonviable. The cell is generally one whichexpresses the antigen to which the protein binds, especially where thecell overexpresses the antigen. Preferably, the cell is a cancer cell,e.g., a breast, ovarian, stomach, endometrial, salivary gland, lung,kidney, colon, thyroid, pancreatic or bladder cell. In vitro, the cellmay be, for example, a SKBR3, BT474, Calu 3, MDA-MB453, MDA-MB-361 orSKOV3 cell. Cell death in vitro may be determined in the absence ofcomplement and immune effector cells to distinguish cell death inducedby antibody dependent cell-mediated cytotoxicity (ADCC) or complementdependent cytotoxicity (CDC). Thus, the assay for cell death may beperformed using heat inactivated serum (i.e., in the absence ofcomplement) and in the absence of immune effector cells. To determinewhether the protein of the invention is able to induce cell death, lossof membrane integrity as evaluated by uptake of propidium iodide (PI),trypan blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD canbe assessed relative to untreated cells.

A protein of the invention that “induces apoptosis” is one that inducesprogrammed cell death as determined by binding of apoptosis relatedmolecules or events, such as annexin V, fragmentation of DNA, cellshrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/orformation of membrane vesicles (called apoptotic bodies). The cell isone which expresses the antigen to which the protein binds and may beone which overexpresses the antigen. The cell may be a tumor cell, e.g.a breast, ovarian, stomach, endometrial, salivary gland, lung, kidney,colon, thyroid, pancreatic or bladder cell. In vitro, the cell may be,for example, SKBR3, BT474, Calu 3 cell, MDA-MB453, MDA-MB-361 or SKOV3cell. Various methods are available for evaluating the cellular eventsassociated with apoptosis. For example, phosphatidyl serine (PS)translocation can be measured by annexin binding; DNA fragmentation canbe evaluated through DNA laddering as disclosed in the example herein;and nuclear/chromatin condensation along with DNA fragmentation can beevaluated by any increase in hypodiploid cells. Preferably, the proteinthat induces apoptosis is one which results in about 2 to 50 fold,preferably about 5 to 50 fold, and most preferably about 10 to 50 fold,induction of annexin binding relative to untreated cell in an annexinbinding assay using cells expressing the antigen to which the protein ofthe invention binds.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thedisorder. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the time todisease progression (TTP) and/or determining the response rates (RR).

The half-life of an amino acid sequence or compound can generally bedefined as the time taken for the serum concentration of the polypeptideto be reduced by 50%, in vivo, for example due to degradation of thesequence or compound and/or clearance or sequestration of the sequenceor compound by natural mechanisms. The half-life can be determined inany manner known per se, such as by pharmacokinetic analysis. Suitabletechniques will be clear to the person skilled in the art, and may forexample generally involve the steps of suitably administering to theprimate a suitable dose of the amino acid sequence or compound to betreated; collecting blood samples or other samples from said primate atregular intervals; determining the level or concentration of the aminoacid sequence or compound of the invention in said blood sample; andcalculating, from (a plot of) the data thus obtained, the time until thelevel or concentration of the amino acid sequence or compound of theinvention has been reduced by 50% compared to the initial level upondosing. Reference is for example made to the standard handbooks, such asKenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook forPharmacists and in Peters et al, Pharmacokinete analysis: A PracticalApproach (1996). Reference is also made to “Pharmacokinetics”, M Gibaldi& D Perron, published by Marcel Dekker, 2nd Rev. edition (1982).

Half-life can be expressed using parameters such as the t½-alpha,t½-beta and the area under the curve (AUC). In the presentspecification, an “increase in half-life” refers to an increase in anyone of these parameters, such as any two of these parameters, oressentially all three these parameters. An “increase in half-life” inparticular refers to an increase in the t½-beta, either with or withoutan increase in the t½-alpha and/or the AUC or both.

The term “EGFR” used herein is equivalent to the term HER-1. The terms“EGFR ligand or EGFR ligands” refers to one or more of the EGFR ligands,such as naturally occurring proteins that bind EGFR.

The term “HER” is used herein to refer to members of the Her family ofreceptors including EGFR, Her-2, Her-3, and Her-4. Preferably, the Herfamily member used in the invention is EGFR.

EGFR Binding Polypeptides

In one aspect, the application provides single domain polypeptides thatbind EGFR. In certain aspects, a single domain polypeptide may compriseat least five to seven beta or beta-like strands distributed among atleast two beta sheets, as exemplified by immunoglobulin andimmunoglobulin-like domains. A beta-like strand is a string of aminoacids that participates in the stabilization of a single domainpolypeptide but does not necessarily adopt a beta strand conformation. Asingle domain polypeptide may comprise between about 80 and about 150amino acids that have a structural organization comprising: at leastseven beta strands or beta-like strands distributed between at least twobeta sheets, and at least one loop portion connecting two beta strandsor beta-like strands, which loop portion participates in binding toEGFR. In other words, a loop portion may link two beta strands, twobeta-like strands or one beta strand and one beta-like strand.Typically, one or more of the loop portions will participate in EGFRbinding, although it is possible that one or more of the beta orbeta-like strand portions will also participate in EGFR binding,particularly those beta or beta-like strand portions that are situatedclosest to the loop portions. In some embodiments, the EGFR bindingpolypeptide inhibits EGFR binding to one or more EGFR ligands. In someembodiments, the EGFR binding polypeptide inhibits EGFR signaling.

In one aspect, the single domain polypeptide comprises an immunoglobulin(Ig) variable domain. The Ig variable domain may, for example, beselected from the group consisting of: a human V_(L) domain, a humanV_(H) domain and a camelid V_(HH) domain. One, two, three or more loopsof the Ig variable domain may participate in binding to EGFR, andtypically any of the loops known as CDR1, CDR2 or CDR3 will participatein EGFR binding.

In one aspect, the single domain polypeptide is a fibronectin basedscaffold protein, i.e., a polypeptide based on a fibronectin type IIIdomain (Fn3). An example of fibronectin-based scaffold proteins areAdnectins™ (Adnexus, a Bristol-Myers Squibb R&D Company). Fibronectin isa large protein which plays essential roles in the formation ofextracellular matrix and cell-cell interactions; it consists of manyrepeats of three types (types I, II, and III) of small domains (Baron etal., 1991). Fn3 itself is the paradigm of a large subfamily whichincludes portions of cell adhesion molecules, cell surface hormone andcytokine receptors, chaperoning, and carbohydrate-binding domains. Forreviews see Bork & Doolittle, Proc Natl Acad Sci USA. 1992 Oct. 1;89(19):8990-4; Bork et al., J Mol Biol. 1994 Sep. 30; 242(4):309-20;Campbell & Spitzfaden, Structure. 1994 May 15; 2(5):333-7; Harpez &Chothia, J Mol Biol. 1994 May 13; 238(4):528-39).

In some embodiments, the application provides fibronectin type III (Fn3)domains that bind EGFR. Such domains may comprise, in order fromN-terminus to C-terminus, a beta or beta-like strand, A; a loop, AB; abeta or beta-like strand, B; a loop, BC; a beta or beta-like strand C; aloop CD; a beta or beta-like strand D; a loop DE; a beta or beta-likestrand, E; a loop, EF; a beta or beta-like strand F; a loop FG; and abeta or beta-like strand G. Any or all of loops AB, BC, CD, DE, EF andFG may participate in EGFR binding. In some embodiments, loops BC and FGparticipate in EGFR binding. In some embodiments, loops BC, DE and FGparticipate in EGFR binding.

In some embodiments, the disclosure provides Fn3 domains having at leastone loop selected from loop BC, DE, and FG with an altered amino acidsequence relative to the sequence of the corresponding loop of the humanFn3 domain. By “altered” is meant one or more amino acid sequencealterations relative to a template sequence (corresponding humanfibronectin domain) and includes amino acid additions, deletions, andsubstitutions. Altering an amino acid sequence may be accomplishedthrough intentional, blind, or spontaneous sequence variation, generallyof a nucleic acid coding sequence, and may occur by any technique, forexample, PCR, error-prone PCR, or chemical DNA synthesis. In someembodiments, the EGFR binding Fn3 domain inhibits EGFR binding to one ormore EGFR ligands. In some embodiments, the EGFR binding Fn3 inhibitsEGFR signaling.

In some embodiments, the Fn3 domain is an Fn3 domain derived from humanfibronectin, particularly the tenth Fn3 domain of fibronectin (¹⁰Fn3),as shown in SEQ ID NO: 1. A variety of mutant ¹⁰Fn3 scaffolds have beenreported. In one aspect, one or more of Asp 7, Glu 9, and Asp 23 isreplaced by another amino acid, such as, for example, a non-negativelycharged amino acid residue (e.g., Asn, Lys, etc.). These mutations havebeen reported to have the effect of promoting greater stability of themutant ¹⁰Fn3 at neutral pH as compared to the wild-type form (See, PCTPublication No. WO02/04523). A variety of additional alterations in the¹⁰Fn3 scaffold that are either beneficial or neutral have beendisclosed. See, for example, Batori et al., Protein Eng. 2002 December;15(12):1015-20; Koide et al., Biochemistry 2001 Aug. 28;40(34):10326-33.

Both variant and wild-type ¹⁰Fn3 proteins are characterized by the samestructure, namely seven beta-strand domain sequences designated Athrough G and six loop regions (AB loop, BC loop, CD loop, DE loop, EFloop, and FG loop) which connect the seven beta-strand domain sequences.The beta strands positioned closest to the N- and C-termini may adopt abeta-like conformation in solution. In SEQ ID NO:1, the AB loopcorresponds to residues 15-16, the BC loop corresponds to residues22-30, the CD loop corresponds to residues 39-45, the DE loopcorresponds to residues 51-55, the EF loop corresponds to residues60-66, and the FG loop corresponds to residues 76-87.

In some embodiments, an EGFR binding ¹⁰Fn3 polypeptide may be at least60%, 65%, 70%, 75%, 80%, 85%, or 90% identical to the human ¹⁰Fn3domain, shown in SEQ ID NO:1. Much of the variability will generallyoccur in one or more of the loops. Each of the beta or beta-like strandsof a ¹⁰Fn3 polypeptide may consist essentially of an amino acid sequencethat is at least 80%, 85%, 90%, 95% or 100% identical to the sequence ofa corresponding beta or beta-like strand of SEQ ID NO: 1, provided thatsuch variation does not disrupt the stability of the polypeptide inphysiological conditions.

In some embodiments, the disclosure provides EGFR binding polypeptidescomprising a tenth fibronectin type III (¹⁰Fn3) domain, wherein the¹⁰Fn3 domain comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; aloop EF; and a loop FG; and has at least one loop selected from loop BC,DE, and FG with an altered amino acid sequence relative to the sequenceof the corresponding loop of the human ¹⁰Fn3 domain. By “altered” ismeant one or more amino acid sequence alterations relative to a templatesequence (corresponding human fibronectin domain) and includes aminoacid additions, deletions, and substitutions. Altering an amino acidsequence may be accomplished through intentional, blind, or spontaneoussequence variation, generally of a nucleic acid coding sequence, and mayoccur by any technique, for example, PCR, error-prone PCR, or chemicalDNA synthesis.

In some embodiments, one or more loops selected from BC, DE, and FG maybe extended or shortened in length relative to the corresponding humanfibronectin loop. In some embodiments, the length of the loop may beextended by from 2-25 amino acids. In some embodiments, the length ofthe loop may be decreased by 1-11 amino acids. In particular, the FGloop of ¹⁰Fn3 is 12 residues long, whereas the corresponding loop inantibody heavy chains ranges from 4-28 residues. To optimize antigenbinding, therefore, the length of the FG loop of ¹⁰Fn3 may be altered inlength as well as in sequence to cover the CDR3 range of 4-28 residuesto obtain the greatest possible flexibility and affinity in antigenbinding. In some embodiments, the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) may be replaced by a polar aminoacid-neutral amino acid-acidic amino acid sequence (in the N-terminal toC-terminal direction).

In some embodiments, the polypeptide comprising a ¹⁰Fn3 domain comprisesthe amino acid sequence of any one of SEQ ID NOS: 207-231. Additionalsequences may be added to the N- or C-terminus. For example, anadditional MG sequence may be placed at the N-terminus. The M willusually be cleaved off, leaving a GVS . . . sequence at the N-terminus.In some embodiments, linker sequences may be placed at the C-terminus ofthe ¹⁰Fn3 domain, e.g., SEQ ID NOS: 233 and 235.

In certain aspects, the disclosure provides short peptide sequences thatmediate EGFR binding. Such sequences may mediate EGFR binding in anisolated form or when inserted into a particular protein structure, suchas an immunoglobulin or immunoglobulin-like domain. Examples of suchsequences include the amino acid residues that correspond to the BC, DE,and FG loops from SEQ ID NOS: 207-231. In some embodiments, the peptidesbind to EGFR with a K_(D) of less than 10⁻⁶M, 10 M, 10 M, 10 M, 10 M orless.

Polypeptide binding to EGFR may be assessed in terms of equilibriumconstants (e.g., dissociation, K_(D)) and in terms of kinetic constants(e.g., on rate constant, k_(on) and off rate constant, k_(off)). Asingle domain polypeptide will typically be selected to bind to EGFRwith a K_(D) of less than 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, 10⁻⁹M or less,although higher K_(D) values may be tolerated where the k_(off) issufficiently low or the k_(on) is sufficiently high. In someembodiments, the EGFR binding polypeptide binds to a related receptor,such as HER2 or HER3, with a K_(D) of more than 10⁻⁶M, 10⁻⁵M, 10⁻⁴M,10⁻³M, 10⁻²M or greater.

In one aspect, the application provides for EGFR binding polypeptidesfurther comprising a pharmacokinetic (PK) moiety. Improvedpharmacokinetics may be assessed according to the perceived therapeuticneed. Often it is desirable to increase bioavailability and/or increasethe time between doses, possibly by increasing the time that a proteinremains available in the serum after dosing. In some instances, it isdesirable to improve the continuity of the serum concentration of theprotein over time (e.g., decrease the difference in serum concentrationof the protein shortly after administration and shortly before the nextadministration). The EGFR binding polypeptides may be attached to amoiety that reduces the clearance rate of the polypeptide in a mammal(e.g., mouse, rat, or human) by greater than three-fold relative to theunmodified polypeptide. Other measures of improved pharmacokinetics mayinclude serum half-life, which is often divided into an alpha phase anda beta phase. Either or both phases may be improved significantly byaddition of an appropriate moiety.

Moieties that tend to slow clearance of a protein from the blood, hereinreferred to as “PK moieties”, include polyoxyalkylene moieties, e.g.,polyethylene glycol, sugars (e.g., sialic acid), and well-toleratedprotein moieties (e.g., Fc, Fc fragments, transferrin, or serumalbumin). The EGFR binding polypeptides may be fused to albumin or afragment (portion) or variant of albumin as described in U.S.Publication No. 20070048282.

In some embodiments, the PK moiety is a serum albumin binding proteinsuch as those described in U.S. Publication Nos. 2007/0178082 and2007/0269422.

In some embodiments, the PK moiety is a serum immunoglobulin bindingprotein such as those described in U.S. Publication No. 2007/0178082.

In some embodiments, the EGFR binding polypeptide comprises polyethyleneglycol (PEG). One or more PEG molecules may be attached at differentpositions on the protein, and such attachment may be achieved byreaction with amines, thiols or other suitable reactive groups. Theamine moiety may be, for example, a primary amine found at theN-terminus of a polypeptide or an amine group present in an amino acid,such as lysine or arginine. In some embodiments, the PEG moiety isattached at a position on the polypeptide selected from the groupconsisting of: a) the N-terminus; b) between the N-terminus and the mostN-terminal beta strand or beta-like strand; c) a loop positioned on aface of the polypeptide opposite the EGFR-binding site; d) between theC-terminus and the most C-terminal beta strand or beta-like strand; ande) at the C-terminus.

Pegylation may be achieved by site-directed pegylation, wherein asuitable reactive group is introduced into the protein to create a sitewhere pegylation preferentially occurs. In some embodiments, the proteinis modified to introduce a cysteine residue at a desired position,permitting site directed pegylation on the cysteine. In someembodiments, the EGFR binding polypeptide comprises a Cys containinglinker such as SEQ ID NO: 235, which permits site directed pegylation.PEG may vary widely in molecular weight and may be branched or linear.

In some embodiments, the EGFR binding polypeptide comprises a Fn3 domainand a PK moiety. In some embodiments, the Fn3 domain is a ¹⁰Fn3 domain.In some embodiments, the PK moiety increases the serum half-life of theEGFR binding polypeptide by more than 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 100, 120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3domain alone.

In some embodiments, the PK moiety is operably linked to the Fn3 domainvia at least one disulfide bond, a peptide bond, a polypeptide, apolymeric sugar, or a polyethylene glycol moiety. Exemplary polypeptidelinkers include PSTSTST (SEQ ID NO: 232), EIDKPSQ (SEQ ID NO: 233), andGS linkers, such as GSGSGSGSGS (SEQ ID NO: 234) and multimers thereof.In some embodiments the PK moiety is human serum albumin. In someembodiments, the PK moiety is transferrin.

In certain aspects, the disclosure provides EGFR binding polypeptidesthat bind to EGFR from a first mammal and to a homolog thereof from asecond mammal. Such polypeptides are particularly useful where the firstmammal is a human and the second mammal is a desirable mammal in whichto conduct preclinical testing, such as a mouse, rat, guinea pig, dog,or non-human primate. In some embodiments, an EGFR binding polypeptidewill bind to both the preselected human target protein and to thehomolog thereof with a K_(D) of less than 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M,10⁻⁹M or less.

EGFR binding polypeptides may bind to any part of EGFR. In someembodiments, the polypeptides bind to an extracellular domain of a EGFR.In some embodiments, the polypeptides bind to the ligand binding domainof EGFR and disrupt the interaction of EGFR with one or more ligands,including TGF-alpha and EGF. In some embodiments, EGFR bindingpolypeptides compete with an anti-EGFR antibody for binding to EGFR. Theanti-EGFR antibody may be selected from any known anti-EGFR antibodyincluding panitumumab (Amgen), nimotuzumab (YM Biosciences), zalutumumab(Genmab), EMD72000 (Merck KGaA), and cetuximab (ImClone Systems)

In some embodiments, the polypeptides bind to EGFR and disrupt receptordimerization. In some embodiments, the EGFR binding polypeptide inhibitsEGFR signaling.

In some embodiments, polypeptide binding to EGFR does not activate EGFRat sub-IC₅₀ concentrations in cell-based assays.

In some embodiments, EGFR binding polypeptides inhibit downstreamsignaling of EGFR. EGFR ligand binding leads to homo- or heterodimericreceptor dimerization with EGFR or another HER family member.Dimerization promotes receptor autophosphorylation, which in turn leadsto the activation of several signaling pathways. One pathway which isactivated is the MAPK pathway, including the phosphorylation of MEK.Another activated pathway is the phosphatidylinositol 3-kinase (PI3K)pathway, including phosphorylation of AKT. Signaling is transduced tothe nucleus, resulting in the activation of various transcriptionfactors. In some embodiments, EGFR binding polypeptides inhibit EGFRligand mediated-EGFR phosphorylation, ERK phosphorylation, AKTphosphorylation, or any other EGFR signaling pathway member with an IC₅₀of less than 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, or 10⁻⁹ M.

Multi-Domain Embodiments

One aspect of the application provides for EGFR binding polypeptidesfurther comprising a second domain. The second domain may bind EGFR or adifferent protein, preferably a human protein. In some embodiments, thesecond domain binds a target selected from FGFR, FGFR1, FGFR2, FGFR3,FGFR4, FGFR5, c-Kit, human p185 receptor-like tyrosine kinase, EGFR,HER2, HER3, HER4, c-Met, folate receptor, PDGFR, VEGFR1, VEGFR2, VEGFR3,human vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, humanCD20, human CD18, human CD11a, human apoptosis receptor-2 (Apo-2), humanalpha4beta7 integrin, human GPIIb-IIIa integrin, stem cell factor (SCF),human CD3, IGF-IR, Ang1, Ang2, fibroblast growth factor, epidermalgrowth factor, hepatocyte growth factor, or Tie2.3. In some embodiments,the second domain binds a human target protein with a K_(D) of less than10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M, or 10⁻⁹M. In some embodiments, the seconddomain binds an protein related to the target protein with a K_(D) ofmore than 10⁻⁶M, 10⁻⁵M, 10⁻⁴M, 10⁻³M, 10⁻²M or greater. In someembodiments, the second domain inhibits the binding target.

One aspect of the application provides an EGFR binding polypeptidefurther comprising a second domain that binds a tumor associated targetor antigen. In some embodiments antigen targeting will help localize theEGFR binding polypeptide in terms of tissue distribution or increasedlocal concentration affect either in the tissue or desired cell type.Alternatively, the second domain may provide an additional mechanism ofaction to combat cancer along with the EGFR binding polypeptide.

In some embodiments, the second domain binds a tumor associated targetor antigen, such as, for example, carbonic anhydrase IX, A3, antigenspecific for A33 antibody, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15,CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80,HLA-DR, NCA 95, NCA90, HCG and its subunits, CEA (CEACAM-5), CEACAM-6,CSAp, EGFR, EGP-1, EGP-2, Ep-CAM, Ba 733, HER2/neu, hypoxia induciblefactor (HIF), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophageinhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, PAM-4-antigen,PSA, PSMA, RS5, S100, TAG-72, p53, tenascin, IL-6, IL-8, insulin growthfactor-I (IGF-I), insulin growth factor-II (IGF-II), Tn antigen,Thomson-Friedenreich antigens, tumor necrosis antigens, placenta growthfactor (PlGF), 17-1A-antigen, an angiogenesis marker (e.g., ED-Bfibronectin), an oncogene marker, an oncogene product, and othertumor-associated antigens. Recent reports, on tumor associated antigensinclude Mizukami et al., (2005, Nature Med. 11:992-97); Hatfield et al.,(2005, Curr. Cancer Drug Targets 5:229-48); Vallbohmer et al. (2005, J.Clin. Oncol. 23:3536-44); and Ren et al. (2005, Ann. Surg. 242:55-63),each incorporated herein by reference.

Any type of tumor and any type of tumor antigen may be targeted with thecorresponding biology of the therapeutic. The cancer can be one or moreof, for example, breast cancer, colon cancer, ovarian carcinoma,osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovialcarcinoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma,and rhabdomyosarcoma, or other cancer yet to be determined in which EGFRlevels are elevated, up-regulated, mutated or altered in physiologycompared to non-oncogenic cells.

Other exemplary types of tumors that may be targeted include acutelymphoblastic leukemia, acute myelogenous leukemia, biliary cancer,breast cancer, cervical cancer, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colorectal cancer, endometrial cancer, esophageal,gastric, head and neck cancer, Hodgkin's lymphoma, lung cancer,medullary thyroid cancer, non-Hodgkin's lymphoma, multiple myeloma,renal cancer, ovarian cancer, pancreatic cancer, glioma, melanoma, livercancer, prostate cancer, and urinary bladder cancer.

In some embodiments, the second domain is selected from an antibodymoiety.

An antibody moiety refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that immunospecificallybinds an antigen. As such, the term antibody moiety encompasses not onlywhole antibody molecules, but also antibody multimers and antibodyfragments as well as variants (including derivatives) of antibodies,antibody multimers and antibody fragments. Examples of antibody moietiesinclude, but are not limited to single chain Fvs (scFvs), Fab fragments,Fab′ fragments, F(ab′)₂, disulfide linked Fvs (sdFvs), and Fvs. Antibodymoieties may be, for example, monoclonal, chimeric, human, or humanized.

In some embodiments, the antibody moiety is selected from (i) a Fabfragment, having VL, CL, VH and CH1 domains; (ii) a Fab′ fragment, whichis a Fab fragment having one or more cysteine residues at the C-terminusof the CH1 domain; (iii) a Fd fragment having VH and CH1 domains; (iv) aFd′ fragment having VH and CH1 domains and one or more cysteine residuesat the C-terminus of the CH1 domain; (v) a Fv fragment having the VL andVH domains of a single arm of an antibody; (vi) a dAb fragment (Ward etal., Nature 341, 544-546 (1989)) which consists of a VH domain; (vii)isolated CDR regions; (viii) F(ab′)₂ fragments, a bivalent fragmentincluding two Fab′ fragments linked by a disulphide bridge at the hingeregion; (ix) single chain antibody molecules (e.g., single chain Fv;scFv) (Bird et al., Science 242:423-426 (1988); and Huston et al., PNAS(USA) 85:5879-5883 (1988)); (x) a “diabody” with two antigen bindingsites, comprising a heavy chain variable domain (VH) connected to alight chain variable domain (VL) in the same polypeptide chain (see,e.g., EP Patent Publication No. 404,097; WO93/11161; and Hollinger etal., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and (xi) a“linear antibody” comprising a pair of tandem Fd segments(VH-CH1-VH-CH1) which, together with complementary light chainpolypeptides, form a pair of antigen binding regions (Zapata et al.Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).

In some embodiments, an antibody moiety is a single domain antibody.Examples include, but are not limited to, heavy chain antibodies,antibodies naturally devoid of light chains, single domain antibodiesderived from conventional 4-chain antibodies, engineered antibodies andsingle domain scaffolds other than those derived from antibodies. Singledomain antibodies may be derived from any species including, but notlimited to mouse, human, camel, llama, goat, rabbit, bovine.

In some embodiments, a single domain antibody is a naturally occurringsingle domain antibody such as VHH domains. VHHs are heavy chainvariable domains derived from immunoglobulins naturally devoid of lightchains such as those derived from Camelidae (including camel, dromedary,llama, vicuna, alpaca and guanaco) as described in WO94/04678. VHHmolecules are about 10 times smaller than IgG molecules. Since VHH's areknown to bind to ‘unusual’ epitopes such as cavities or grooves, theaffinity of such VHH's may be more suitable for therapeutic treatment,PCT Publication No. WO97/49805.

In some embodiments, the single domain antibody is a VHH that binds aserum protein as described in U.S. Publication No. 20070178082. Theserum protein may be any suitable protein found in the serum of subject,or fragment thereof. In some embodiments, the serum protein is serumalbumin, serum immunoglobulins, thyroxine-binding protein, transferrin,or fibrinogen.

Various techniques have been developed for the production of antibodyfragments that may be used to make antibody fragments used in theinvention. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. For example, the antibodyfragments can be isolated from the antibody phage libraries discussedabove. Alternatively, Fab′-SH fragments can be directly recovered fromE. coli and chemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-167 (1992)). According to another approach,F(ab′)₂ fragments can be isolated directly from recombinant host cellculture. Other techniques for the production of antibody fragments willbe apparent to the skilled practitioner. In other embodiments, theantibody of choice is a single chain Fv fragment (scFv). See WO93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458. Theantibody fragment may also be a “linear antibody”, e.g., as described inU.S. Pat. No. 5,641,870 for example. Such linear antibody fragments maybe monospecific or bispecific.

In some embodiments, the second domain comprises one or more avimersequences. Avimers were developed from human extracellular receptordomains by in vitro exon shuffling and phage display. (Silverman et al.,2005, Nat. Biotechnol. 23:1493-94; Silverman et al., 2006, Nat.Biotechnol. 24:220.) The resulting multidomain proteins may comprisemultiple independent binding domains that may exhibit improved affinity(in some cases sub-nanomolar) and specificity compared withsingle-epitope binding proteins. Additional details concerning methodsof construction and use of avimers are disclosed, for example, in U.S.Patent Publication Nos. 20040175756, 20050048512, 20050053973,20050089932 and 20050221384, which is incorporated herein by referencein their entirety.

In some embodiments, the second domain comprises one or more lipocalinrelated sequences, e.g., anticalins or lipocalin derivatives. Anticalinsor lipocalin derivatives are a type of binding proteins that haveaffinities and specificities for various target molecules, includingthose described herein. Such proteins are described in US PatentPublication Nos. 20060058510, 20060088908, 20050106660, and PCTPublication No. WO2006/056464.

In some embodiments, the second domain comprises one or more tetranectinC-type lectin related sequences or trinectins, e.g., tetranectin C-typelectin or tetranectin C-type lectin derivatives. Tetranectin C-typelectins or tetranectin C-type lectin derivatives are a type of bindingproteins that have affinities and specificities for various targetmolecules including those described herein. Different tetranectin C-typelectin and related proteins are described in PCT Publication Nos.WO2006/053568, WO2005/080418, WO2004/094478, WO2004/039841,WO2004/005335, WO2002/048189, WO98/056906, and U.S. Patent PublicationNo. 20050202043.

In some embodiments, the second domain comprises one or more naturalankyrin repeat proteins, e.g., DARPins (Molecular Partners).

In some embodiments, the second domain comprises one or moreAffibodies™. Affibodies™ are derived from the IgG binding domain ofStaphyloccal Protein A. Novel binding properties can be achieved byaltering residues located near the binding surface of the Protein Adomain. In some embodiments, the second domain comprises one or morecystein knot based protein scaffolds, i.e., microbodies(Selecore/NascaCell).

In some embodiments, the second domain comprises one or moreTrans-bodies™. Trans-bodies™ are based on transferrin scaffolds(BioResis/Pfizer).

In some embodiments, the second domain comprises binding proteins basedon gamma-crystallin or ubiquitin. These so-called Affilin™ (ScilProteins) molecules are characterized by the de novo design of a bindingregion in beta sheet structures of the proteins. Affilin™ molecules havebeen described in U.S Publication No. 20070248536.

In some embodiments, the second domain comprises a Fn3 domain. In someembodiments, the Fn3 domain is an Fn3 domain derived from humanfibronectin, particularly the tenth Fn3 domain of fibronectin (¹⁰Fn3).In some embodiments, the EGFR binding polypeptide is a Fn3 domain.

In some embodiments, one or more loops of the Fn3 domain selected fromBC, DE, and FG may be extended or shortened in length relative to thecorresponding human fibronectin loop. In some embodiments, the length ofthe loop may be extended by from 2-25 amino acids. In some embodiments,the integrin-binding motif “arginine-glycine-aspartic acid” (RGD) may bereplaced by a polar amino acid-neutral amino acid-acidic amino acidsequence (in the N-terminal to C-terminal direction).

In some embodiments, the Fn3 domain binds human IGF-IR, EGFR, or VEGFR2.In some embodiments, the Fn3 domain binds human IGF-IR, comprises theamino acid sequence of any one of SEQ ID NOS: 2-125, 184-204, 236, andinhibits IGF-IR signaling. In some embodiments, the Fn3 domain bindshuman EGFR, comprises the amino acid sequence of any one of SEQ ID NOS:207-231, and inhibits EGFR signaling. In some embodiments, the Fn3domain binds human VEGFR2, comprises the amino acid sequence of any oneof SEQ ID NOS: 126-183, 205, 206, and inhibits VEGFR2 signaling. In someembodiments, the Fn3 domain is a ¹⁰Fn3 domain comprising the amino acidsequence of any one of SEQ ID NOS: 2-231 or 236. In some embodiments,the ¹⁰Fn3 domain comprises an amino acid sequence at least 75, 80, 85,90, 95, or 98% identical to any one of SEQ ID NOS: 2-231 or 236.

Conjugation

One aspect of the application provides polypeptides comprising an EGFRbinding polypeptide and a second domain operably linked via at least onedisulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or aPEG moiety.

In some embodiments, the EGFR binding polypeptide and the second domainare operably linked via a polypeptide. In some embodiments, thepolypeptide linker is SEQ ID NOS: 233 or 235.

In some embodiments, the EGFR binding polypeptide and the second domainare operably linked via a polypeptide linker having a protease site thatis cleavable by a protease in the blood or target tissue. Suchembodiments can be used to release two or more therapeutic proteins forbetter delivery or therapeutic properties or more efficient productioncompared to separately producing such proteins.

In some embodiments, the EGFR binding polypeptide and the second domainare operably linked via a biocompatible polymer such as a polymericsugar. Such polymeric sugar can include an enzymatic cleavage site thatis cleavable by an enzyme in the blood or target tissue. Suchembodiments can be used to release two or more therapeutic proteins forbetter delivery or therapeutic properties or more efficient productioncompared to separately producing such proteins.

In some embodiments, the EGFR binding polypeptide and the second domainare operably linked via a polymeric linker. Polymeric linkers can beused to optimally vary the distance between each protein moiety tocreate a protein with one or more of the following characteristics: 1)reduced or increased steric hindrance of binding of one or more proteindomain when binding to a protein of interest, 2) increased proteinstability or solubility without searching for additional amino acidsubstitutions to increase stability or solubility (e.g., solubility atleast about 20 mg/ml, or at least about 50 μg/ml), 3) decreased proteinaggregation without searching for additional amino acid substitutions todecrease stability (e.g., as measured by SEC), and 4) increased theoverall avidity or affinity of the protein by adding additional bindingdomains.

In some embodiments, the EGFR binding polypeptide is a ¹⁰Fn3 domaincomprising the linker of SEQ ID NO: 235. PEG is conjugated to thecysteine moiety in the linker sequence and operably links the EGFRbinding polypeptide to a second domain.

PEGylated Embodiments

One aspect of the application provides linking the EGFR bindingpolypeptides to nonproteinaceous polymers. In some embodiments, thepolymer is polyethylene glycol (“PEG”), polypropylene glycol, orpolyoxyalkylenes, as described in U.S. Pat. No. 4,640,835; 4,496,689;4,301,144; 4,670,417; 4,791,192 or 4,179,337. In some embodiments, theEGFR binding polypeptides comprise an Fn3 domain. In some embodiments,the polymer is a PEG moiety. In addition, the application provides N orC terminal PEG conjugation to antibody moieties (e.g., camel antibodiesand their derivatives, as well as single chain and domain antibodies;and particularly those expressed from microbes) and antibody-likemoieties (e.g., derivatives of lipocalins, ankyrins, multiple Cys-Cysdomains, and tetranectins; and particularly those expressed frommicrobes).

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented by the formula: X—O(CH₂CH₂O)_(n-1)CH₂CH₂OH(1), where n is 20 to 2300 and X is H or a terminal modification, e.g.,a C₁₋₄ alkyl. In one embodiment, the PEG of the invention terminates onone end with hydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). APEG can contain further chemical groups which are necessary for bindingreactions; which results from the chemical synthesis of the molecule; orwhich is a spacer for optimal distance of parts of the molecule. Inaddition, such a PEG can consist of one or more PEG side-chains whichare linked together. PEGs with more than one PEG chain are calledmultiarmed or branched PEGs. Branched PEGs can be prepared, for example,by the addition of polyethylene oxide to various polyols, includingglycerol, pentaerythriol, and sorbitol. For example, a four-armedbranched PEG can be prepared from pentaerythriol and ethylene oxide.Branched PEG are described in, for example, European PublishedApplication No. 473084A and U.S. Pat. No. 5,932,462. One form of PEGsincludes two PEG side-chains (PEG2) linked via the primary amino groupsof a lysine (Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).

PEG conjugation to peptides or proteins generally involves theactivation of PEG and coupling of the activated PEG-intermediatesdirectly to target proteins/peptides or to a linker, which issubsequently activated and coupled to target proteins/peptides (seeAbuchowski, A. et al, J. Biol. Chem., 252, 3571 (1977) and J. Biol.Chem., 252, 3582 (1977), Zalipsky, et al., and Harris et. al., in:Poly(ethylene glycol) Chemistry: Biotechnical and BiomedicalApplications; (J. M. Harris ed.) Plenum Press: New York, 1992; Chap. 21and 22). It is noted that a binding polypeptide containing a PEGmolecule is also known as a conjugated protein, whereas the proteinlacking an attached PEG molecule can be referred to as unconjugated.

The size of PEG utilized will depend on several factors including theintended use of the EGFR binding polypeptide. Larger PEGs are preferredto increase half life in the body, blood, non-blood extracellular fluidsor tissues. For in vivo cellular activity, PEGs of the range of about 10to 60 kDa are preferred, as well as PEGs less than about 100 kDa andmore preferably less than about 60 kDa, though sizes greater than about100 kDa can be used as well. For in vivo imaging application, smallerPEGs, generally less than about 20 kDa, may be used that do not increasehalf life as much as larger PEGs so as to permit quicker distributionand less half life. A variety of molecular mass forms of PEG can beselected, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20 to2300), for conjugating to binding polypeptides of the invention. Thenumber of repeating units “n” in the PEG is approximated for themolecular mass described in Daltons. It is preferred that the combinedmolecular mass of PEG on an activated linker is suitable forpharmaceutical use. Thus, in one embodiment, the molecular mass of thePEG molecules does not exceed 100,000 Da. For example, if three PEGmolecules are attached to a linker, where each PEG molecule has the samemolecular mass of 12,000 Da (each n is about 270), then the totalmolecular mass of PEG on the linker is about 36,000 Da (total n is about820). The molecular masses of the PEG attached to the linker can also bedifferent, e.g., of three molecules on a linker two PEG molecules can be5,000 Da each (each n is about 110) and one PEG molecule can be 12,000Da (n is about 270). In some embodiments, one PEG moiety is conjugatedto the EGFR binding polypeptide. In some embodiments, the PEG moiety isabout 30, 40, 50, 60, 70, 80, or 90 KDa.

In some embodiments, PEGylated EGFR binding polypeptides contain one,two or more PEG moieties. In one embodiment, the PEG moiety(ies) arebound to an amino acid residue which is on the surface of the proteinand/or away from the surface that contacts the target ligand. In oneembodiment, the combined or total molecular mass of PEG in PEG-bindingpolypeptide is from about 3,000 Da to 60,000 Da, or from about 10,000 Dato 36,000 Da. In a one embodiment, the PEG in pegylated bindingpolypeptide is a substantially linear, straight-chain PEG.

One skilled in the art can select a suitable molecular mass for PEG,e.g., based on how the pegylated binding polypeptide will be usedtherapeutically, the desired dosage, circulation time, resistance toproteolysis, immunogenicity, and other considerations. For a discussionof PEG and its use to enhance the properties of proteins, see N. V.Katre, Advanced Drug Delivery Reviews 10: 91-114 (1993).

In some embodiments, an EGFR binding polypeptide is covalently linked toone poly(ethylene glycol) group of the formula:—CO—(CH₂)_(x)—(OCH₂CH₂)_(m)—OR, with the —CO (i.e. carbonyl) of thepoly(ethylene glycol) group forming an amide bond with one of the aminogroups of the binding polypeptide; R being lower alkyl; x being 2 or 3;m being from about 450 to about 950; and n and m being chosen so thatthe molecular weight of the conjugate minus the binding polypeptide isfrom about 10 to 40 kDa. In one embodiment, a binding polypeptide'sε-amino group of a lysine is the available (free) amino group.

In one specific embodiment, carbonate esters of PEG are used to form thePEG-binding polypeptide conjugates. N,N′-disuccinimidylcarbonate (DSC)may be used in the reaction with PEG to form active mixedPEG-succinimidyl carbonate that may be subsequently reacted with anucleophilic group of a linker or an amino group of a bindingpolypeptide (see U.S. Pat. No. 5,281,698 and U.S. Pat. No. 5,932,462).In a similar type of reaction, 1,1′-(dibenzotriazolyl)carbonate anddi-(2-pyridyl)carbonate may be reacted with PEG to formPEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.5,382,657), respectively.

Pegylation of an EGFR binding polypeptide can be performed according tothe methods of the state of the art, for example by reaction of thebinding polypeptide with electrophilically active PEGs (supplier:Shearwater Corp., USA, www.shearwatercorp.com). Preferred PEG reagentsof the present invention are, e.g., N-hydroxysuccinimidyl propionates(PEG-SPA), butanoates (PEG-SBA), PEG-succinimidyl propionate or branchedN-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to pegylatedat an ε-amino group of a binding polypeptide lysine or the N-terminalamino group of the binding polypeptide.

In another embodiment, PEG molecules may be coupled to sulfhydryl groupson a binding polypeptide (Sartore, L., et al., Appl. Biochem.Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7, 363-368(1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S. Pat. No.5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897 describes exemplaryreactive PEG species that may be coupled to sulfhydryl groups.

In some embodiments, the pegylated an EGFR binding polypeptide isproduced by site-directed pegylation, particularly by conjugation of PEGto a cysteine moiety at the N- or C-terminus. In some embodiments, theEGFR binding polypeptide is a Fn3 domain covalently bound to a PEGmoiety, wherein at least one of the loops of said Fn3 domainparticipates in EGFR binding. The PEG moiety may be attached to the Fn3polypeptide by site directed pegylation, such as by attachment to a Cysresidue, where the Cys residue may be positioned at the N-terminus ofthe Fn3 polypeptide or between the N-terminus and the most N-terminalbeta or beta-like strand or at the C-terminus of the Fn3 polypeptide orbetween the C-terminus and the most C-terminal beta or beta-like strand.A Cys residue may be situated at other positions as well, particularlyany of the loops that do not participate in target binding. A PEG moietymay also be attached by other chemistry, including by conjugation toamines.

In some embodiments where PEG molecules are conjugated to cysteineresidues on a binding polypeptide, the cysteine residues are native tothe binding polypeptide, whereas in other embodiments, one or morecysteine residues are engineered into the binding polypeptide. Mutationsmay be introduced into an binding polypeptide coding sequence togenerate cysteine residues. This might be achieved, for example, bymutating one or more amino acid residues to cysteine. Preferred aminoacids for mutating to a cysteine residue include serine, threonine,alanine and other hydrophilic residues. Preferably, the residue to bemutated to cysteine is a surface-exposed residue. Algorithms arewell-known in the art for predicting surface accessibility of residuesbased on primary sequence or a protein. Alternatively, surface residuesmay be predicted by comparing the amino acid sequences of bindingpolypeptides, given that the crystal structure of the framework based onwhich binding polypeptides are designed and evolved has been solved (seeHimanen et al., Nature. (2001) 20-27; 414(6866):933-8) and thus thesurface-exposed residues identified. In one embodiment, cysteineresidues are introduced into binding polypeptides at or near the N-and/or C-terminus, or within loop regions. Pegylation of cysteineresidues may be carried out using, for example, PEG-maleimide,PEG-vinylsulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

In some embodiments, the pegylated binding polypeptide comprises a PEGmolecule covalently attached to the alpha amino group of the N-terminalamino acid. Site specific N-terminal reductive amination is described inPepinsky et al., (2001) JPET, 297,1059, and U.S. Pat. No. 5,824,784. Theuse of a PEG-aldehyde for the reductive amination of a protein utilizingother available nucleophilic amino groups is described in U.S. Pat. No.4,002,531, in Wieder et al., (1979) J. Biol. Chem. 254,12579, and inChamow et al., (1994) Bioconjugate Chem. 5, 133.

In another embodiment, pegylated binding polypeptide comprises one ormore PEG molecules covalently attached to a linker, which in turn isattached to the alpha amino group of the amino acid residue at theN-terminus of the binding polypeptide. Such an approach is disclosed inU.S. Publication No. 2002/0044921 and PCT Publication No. WO94/01451.

In one embodiment, a binding polypeptide is pegylated at the C-terminus.In a specific embodiment, a protein is pegylated at the C-terminus bythe introduction of C-terminal azido-methionine and the subsequentconjugation of a methyl-PEG-triarylphosphine compound via the Staudingerreaction. This C-terminal conjugation method is described in Cazalis etal., C-Terminal Site-Specific PEGylation of a Truncated ThrombomodulinMutant with Retention of Full Bioactivity, Bioconjug Chem. 2004;15(5):1005-1009.

Conventional separation and purification techniques known in the art canbe used to purify PEGylated binding polypeptide, such as size exclusion(e.g., gel filtration) and ion exchange chromatography. Products mayalso be separated using SDS-PAGE. Products that may be separated includemono-, di-, tri-poly- and un-pegylated binding polypeptide, as well asfree PEG. The percentage of mono-PEG conjugates can be controlled bypooling broader fractions around the elution peak to increase thepercentage of mono-PEG in the composition. About ninety percent mono-PEGconjugates represents a good balance of yield and activity. Compositionsin which, for example, at least ninety-two percent or at leastninety-six percent of the conjugates are mono-PEG species may bedesired. In an embodiment of this invention the percentage of mono-PEGconjugates is from ninety percent to ninety-six percent.

In one embodiment of the invention, the PEG in a pegylated EGFR bindingpolypeptide is not hydrolyzed from the pegylated amino acid residueusing a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH 6.5) over 8to 16 hours at room temperature, and is thus stable. In one embodiment,greater than 80% of the composition is stable mono-PEG-bindingpolypeptide, more preferably at least 90%, and most preferably at least95%.

In another embodiment, the pegylated EGFR binding polypeptides willpreferably retain at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%or 100% of the biological activity associated with the unmodifiedprotein. In one embodiment, biological activity refers to its ability tobind to EGFR, as assessed by K_(D), k_(on) or k_(off). In one specificembodiment, the pegylated binding polypeptide protein shows an increasein binding to EGFR relative to unpegylated binding polypeptide.

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Deimmunization of EGFR Binding Polypeptides

In one aspect, the application provides deimmunized EGFR bindingpolypeptides. In some embodiments, the sequence of an EGFR bindingpolypeptide has been altered to eliminate one or more B- or T-cellepitopes. In some embodiments, the EGFR binding polypeptide comprises an¹⁰Fn3 domain.

The EGFR binding polypeptide may be deimmunized to render itnon-immunogenic, or less immunogenic, to a given species. Deimmunizationcan be achieved through structural alterations to the polypeptide. Anydeimmunization technique known to those skilled in the art can beemployed. One suitable technique, for example, for deimmunizing proteinsis described in WO 00/34317, the disclosure of which is incorporatedherein in its entirety. In summary, a typical protocol within thegeneral method described therein includes the following steps.

1. Determining the amino acid sequence of the polypeptide;2. Identifying potential T-cell epitopes within the amino acid sequenceof the polypeptide by any method including determination of the bindingof peptides to MHC molecules, determination of the binding of peptide:HLA complexes to the T-cell receptors from the species to receive thetherapeutic protein, testing of the polypeptide or parts thereof usingtransgenic animals with HLA molecules of the species to receive thetherapeutic protein, or testing such transgenic animals reconstitutedwith immune system cells from the species to receive the therapeuticprotein;3. By genetic engineering or other methods for producing modifiedpolypeptide, altering the polypeptide to remove one or more of thepotential T-cell epitopes and producing such an altered polypeptide fortesting.

In one embodiment, the sequences of the polypeptide can be analyzed forthe presence of MHC class II binding motifs. For example, a comparisonmay be made with databases of MHC-binding motifs such as, for example bysearching the “motifs” database on the worldwide web atsitewehil.wehi.edu.au. Alternatively, MHC class II binding peptides maybe identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)) wherebyconsecutive overlapping peptides from the polypeptide are testing fortheir binding energies to MHC class II proteins. Computational bindingprediction algorithms include iTope™, Tepitope, SYFPEITHI, EpiMatrix(EpiVax), and MHCpred. In order to assist the identification of MHCclass II-binding peptides, associated sequence features which relate tosuccessfully presented peptides such as amphipathicity and Rothbardmotifs, and cleavage sites for cathepsin B and other processing enzymescan be searched for.

Having identified potential (e.g. human) T-cell epitopes, these epitopesare then eliminated by alteration of one or more amino acids, asrequired to eliminate the T-cell epitope. Usually, this will involvealteration of one or more amino acids within the T-cell epitope itself.This could involve altering an amino acid adjacent the epitope in termsof the primary structure of the protein or one which is not adjacent inthe primary structure but is adjacent in the secondary structure of themolecule. The usual alteration contemplated will be amino acidsubstitution, but it is possible that in certain circumstances aminoacid addition or deletion will be appropriate. All alterations can beaccomplished by recombinant DNA technology, so that the final moleculemay be prepared by expression from a recombinant host, for example bywell established methods, but the use of protein chemistry or any othermeans of molecular alteration may also be used.

Once identified T-cell epitopes are removed, the deimmunized sequencemay be analyzed again to ensure that new T-cell epitopes have not beencreated and, if they have, the epitope(s) can be deleted.

Not all T-cell epitopes identified computationally need to be removed. Aperson skilled in the art will appreciate the significance of the“strength” or rather potential immunogenicity of particular epitopes.The various computational methods generate scores for potentialepitopes. A person skilled in the art will recognize that only the highscoring epitopes may need to be removed. A skilled person will alsorecognize that there is a balance between removing potential epitopesand maintaining binding affinity of the polypeptide. Therefore, onestrategy is to sequentially introduce substitutions into the polypeptideand then test for antigen binding and immunogenicity.

In one aspect the deimmunized polypeptide is less immunogenic (orrather, elicits a reduced HAMA response) than the original polypeptidein a human subject. Assays to determine immunogenicity are well withinthe knowledge of the skilled person. Art-recognized methods ofdetermining immune response can be performed to monitor a HAMA responsein a particular subject or during clinical trials. Subjects administereddeimmunized polypeptide can be given an immunogenicity assessment at thebeginning and throughout the administration of said therapy. The HAMAresponse is measured, for example, by detecting antibodies to thedeimmunized polypeptide, in serum samples from the subject using amethod known to one in the art, including surface plasmon resonancetechnology (BIACORE) and/or solid-phase ELISA analysis. Alternatively,in vitro assays designed to measure a T-cell activation event are alsoindicative of immunogenicity.

Additional Modifications

In some embodiments, the EGFR binding polypeptides are glycosylated. Insome embodiments, the polypeptides are Fn3 domains. Fn3 domains do notnormally contain glycosylation sites, however, such glycosylation may beengineered into the protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.These can be engineered into the proteins of the invention, inparticular fibronectin-based scaffold proteins and their correspondingpolynucleotides. Thus, the presence of either of these tripeptidesequences in a polypeptide creates a potential glycosylation site.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to proteins is conveniently accomplishedby altering the amino acid sequence such that it contains one or more ofthe above-described tripeptide sequences (for N-linked glycosylationsites). The alteration may also be made by the addition of, orsubstitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

In some embodiments, the EGFR binding polypeptides are modified toenhance antigen-dependent cell-mediated cytotoxicity (A DCC) and/orcomplement dependent cytotoxicity (CDC). In some embodiments, the EGFRbinding polypeptide is an Fn3 domain further comprising an Fc region. Insome embodiments, the Fc region is a variant that enhances ADCC or CDC.The Fc region variant may comprise a human Fc region sequence (e.g., ahuman IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acidmodification (e.g., a substitution) at one or more amino acid positions.

In one embodiment, the variant Fc region may mediate antibody-dependentcell-mediated cytotoxicity (ADCC) in the presence of human effectorcells more effectively, or bind an Fc gamma receptor (FcγR) with betteraffinity, than a native sequence Fc region. Such Fc region variants maycomprise an amino acid modification at any one or more of positions 256,290, 298, 312, 326, 330, 333, 334, 360, 378 or 430 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex as in Kabat.

Nucleic Acid-Protein Fusion Technology

In one aspect, the application provides fibronectin type III domainsthat bind a human target, such as, for example, EGFR, VEGFR2, IGF-IR,and other proteins. One way to rapidly make and test Fn3 domains withspecific binding properties is the nucleic acid-protein fusiontechnology of Adnexus, a Bristol-Myers Squibb R&D Company. Thisdisclosure describes the use of such in vitro expression and taggingtechnology, termed PROfusion™, that exploits nucleic acid-proteinfusions (RNA- and DNA-protein fusions) to identify novel polypeptidesand amino acid motifs that are important for binding to EGFR and otherproteins. Nucleic acid-protein fusion technology is a technology thatcovalently couples a protein to its encoding genetic information. For adetailed description of the RNA-protein fusion technology andfibronectin-based scaffold protein library screening methods see Szostaket al., U.S. Pat. Nos. 6,258,558; 6,261,804; 6,214,553; 6,281,344;6,207,446; 6,518,018; PCT Publication Nos. WO00/34784; WO01/64942;WO02/032925; and Roberts and Szostak, Proc Natl. Acad. Sci.94:12297-12302, 1997, herein incorporated by reference. Furtherdiscussion of nucleic acid-protein fusion technology can be found in theExamples and the Material and Methods section of the application.

Vectors & Polynucleotides Embodiments

Nucleic acids encoding any of the various proteins or polypeptidesdisclosed herein may be synthesized chemically. Codon usage may beselected so as to improve expression in a cell. Such codon usage willdepend on the cell type selected. Specialized codon usage patterns havebeen developed for E. coli and other bacteria, as well as mammaliancells, plant cells, yeast cells and insect cells. See for example:Mayfield et al., Proc Natl Acad Sci USA. 2003 Jan. 21; 100(2):438-42;Sinclair et al. Protein Expr Purif. 2002 October; 26(1):96-105; ConnellN D. Curr Opin Biotechnol. 2001 October; 12(5):446-9; Makrides et al.Microbiol Rev. 1996 September; 60(3):512-38; and Sharp et al. Yeast.1991 October; 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The proteins described herein may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The heterologous signal sequence selected preferably isone that is recognized and processed (i.e., cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process a native signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence selected, for example, fromthe group of the alkaline phosphatase, penicillinase, 1 pp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,a factor leader (including Saccharomyces and Kluyveromyces alpha-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in PCT Publication No. WO90/13646. Inmammalian cell expression, mammalian signal sequences as well as viralsecretory leaders, for example, the herpes simplex gD signal, areavailable. The DNA for such precursor regions may be ligated in readingframe to DNA encoding the protein.

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2 micron plasmid origin is suitablefor yeast, and various viral origins (SV40, polyoma, adenovirus, VSV orBPV) are useful for cloning vectors in mammalian cells. Generally, theorigin of replication component is not needed for mammalian expressionvectors (the SV40 origin may typically be used only because it containsthe early promoter).

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the protein of the invention, e.g., a fibronectin-basedscaffold protein. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, beta-lactamase and lactose promoter systems,alkaline phosphatase, a tryptophan (trp) promoter system, and hybridpromoters such as the tac promoter. However, other known bacterialpromoters are suitable. Promoters for use in bacterial systems also willcontain a Shine-Dalgarno (S.D.) sequence operably linked to the DNAencoding the protein of the invention.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP Patent Publication No. 73,657. Yeast enhancers also areadvantageously used with yeast promoters.

Transcription from vectors in mammalian host cells can be controlled,for example, by promoters obtained from the genomes of viruses such aspolyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovinepapilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus,hepatitis-B virus and most preferably Simian Virus 40 (SV40), fromheterologous mammalian promoters, e.g., the actin promoter or animmunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human.beta.-interferon cDNA in mouse cells under thecontrol of a thymidine kinase promoter from herpes simplex virus.Alternatively, the rous sarcoma virus long terminal repeat can be usedas the promoter.

Transcription of a DNA encoding proteins of the invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, .alpha.-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to themultivalent antibody-encoding sequence, but is preferably located at asite 5′ from the promoter.

Expression vectors used in eukaryotic host cells (e.g., yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding the multivalent antibody.One useful transcription termination component is the bovine growthhormone polyadenylation region. See WO94/11026 and the expression vectordisclosed therein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, New York,1985), the relevant disclosure of which is hereby incorporated byreference.

The expression construct is introduced into the host cell using a methodappropriate to the host cell, as will be apparent to one of skill in theart. A variety of methods for introducing nucleic acids into host cellsare known in the art, including, but not limited to, electroporation;transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (where the vector is aninfectious agent).

Suitable host cells include prokaryotes, yeast, mammalian cells, orbacterial cells. Suitable bacteria include gram negative or grampositive organisms, for example, E. coli or Bacillus spp. Yeast,preferably from the Saccharomyces species, such as S. cerevisiae, mayalso be used for production of polypeptides. Various mammalian or insectcell culture systems can also be employed to express recombinantproteins. Baculovirus systems for production of heterologous proteins ininsect cells are reviewed by Luckow and Summers, (Bio/Technology, 6:47,1988). Examples of suitable mammalian host cell lines includeendothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3,Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293,293T, and BHK cell lines. Purified polypeptides are prepared byculturing suitable host/vector systems to express the recombinantproteins. For many applications, the small size of many of thepolypeptides disclosed herein would make expression in E. coli as thepreferred method for expression. The protein is then purified fromculture media or cell extracts.

Suitable host cells for the expression of glycosylated proteins of theinvention are derived from multicellular organisms. Examples ofinvertebrate cells include plant and insect cells. Numerous baculoviralstrains and variants and corresponding permissive insect host cells fromhosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to thepresent invention, particularly for transfection of Spodopterafrugiperda cells.

In some instance it will be desired to produce proteins in vertebratecells, such as for glycosylation, and propagation of vertebrate cells inculture (tissue culture) has become a routine procedure. Examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59. (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; ahuman hepatoma line (Hep G2); and myeloma or lymphoma cells (e.g., Y0,J558L, P3 and NS0 cells) (see U.S. Pat. No. 5,807,715). Plant cellcultures of cotton, corn, potato, soybean, petunia, tomato, and tobaccocan also be utilized as hosts.

Protein Production

Host cells are transformed with the herein-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce the proteins of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S. Pat. No. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

Proteins of the invention can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The proteins of the present invention can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Imaging, Diagnostic and Other Applications

In one aspect, the application provides EGFR binding polypeptideslabeled with a detectable moiety. The polypeptides may be used for avariety of diagnostic applications. The detectable moiety can be any onewhich is capable of producing, either directly or indirectly, adetectable signal. For example, the detectable moiety may be aradioisotope, such as H3, C14 or 13, P32, S35, or 1131; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating a protein to the detectablemoiety may be employed, including those methods described by Hunter, etal., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974);Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem.and Cytochem. 30:407 (1982). In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a moiety (such as PEG) to a protein of theinvention, a linking group or reactive group is used. Suitable linkinggroups are well known in the art and include disulfide groups, thioethergroups, acid labile groups, photolabile groups, peptidase labile groupsand esterase labile groups. Preferred linking groups are disulfidegroups and thioether groups depending on the application. Forpolypeptides without a Cys amino acid, a Cys can be engineered in alocation to allow for activity of the protein to exist while creating alocation for conjugation.

EGFR binding polypeptides linked with a detectable moiety also areuseful for in vivo imaging. The polypeptide may be linked to aradio-opaque agent or radioisotope, administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled protein in the subject is assayed. This imaging technique isuseful in the staging and treatment of malignancies. The protein may belabeled with any moiety that is detectable in a subject, whether bynuclear magnetic resonance, radiology, or other detection means known inthe art.

EGFR binding polypeptides also are useful as affinity purificationagents. In this process, the polypeptides are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art.

EGFR binding polypeptides can be employed in any known assay method,such as competitive binding assays, direct and indirect sandwich assays,and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual ofTechniques, pp. 147-158 (CRC Press, Inc., 1987)).

In certain aspects, the disclosure provides methods for detecting EGFRin a sample. A method may comprise contacting the sample with a EGFRbinding polypeptide described herein, wherein said contacting is carriedout under conditions that allow polypeptide-EGFR complex formation; anddetecting said complex, thereby detecting said EGFR in said sample.Detection may be carried out using any technique known in the art, suchas, for example, radiography, immunological assay, fluorescencedetection, mass spectroscopy, or surface plasmon resonance. The samplewill often by a biological sample, such as a biopsy, and particularly abiopsy of a tumor, a suspected tumor. The sample may be from a human orother mammal. The EGFR binding polypeptide may be labeled with alabeling moiety, such as a radioactive moiety, a fluorescent moiety, achromogenic moiety, a chemiluminescent moiety, or a hapten moiety. TheEGFR binding polypeptide may be immobilized on a solid support.

Therapeutic/In Vivo Uses

In one aspect, the application provides EGFR binding polypeptides usefulin the treatment of EGFR related disorders. The application alsoprovides methods for administering EGFR binding polypeptides to asubject. In some embodiments, the subject is a human. In someembodiments, the subject has an EGFR related disorder, such as cancer.In some embodiments, the EGFR binding polypeptide inhibits EGFRsignaling. In some embodiments, the EGFR binding polypeptide inhibitsEGFR binding to one or more EGFR ligands.

In some embodiments, administration of EGFR binding polypeptides to ananimal causes a reduction in EGFR levels in EGFR-expressing tumors. Insome embodiments, the polypeptide causes a reduction in receptor levelsby at least 20, 30, 40, 50, 60, 70, 80% or more compared to an untreatedanimal.

In some embodiments, administration of an EGFR binding polypeptideinhibits tumor cell growth in vivo. The tumor cell may be derived fromany cell type including, without limitation, epidermal, epithelial,endothelial, leukemia, sarcoma, multiple myeloma, or mesodermal cells.Examples of common tumor cell lines for use in xenograft tumor studiesinclude A549 (non-small cell lung carcinoma) cells, DU-145 (prostate)cells, MCF-7 (breast) cells, Colo 205 (colon) cells, 3T3/]GF-IR (mousefibroblast) cells, NCI H441 cells, HEP G2 (hepatoma) cells, MDA MB 231(breast) cells, HT-29 (colon) cells, MDA-MB-435s (breast) cells, U266cells, SH-SY5Y cells, Sk-MeI-2 cells, NCI-H929, RPM18226, and A431cells. In some embodiments, the polypeptide inhibits tumor cell growthrelative to the growth of the tumor in an untreated animal. In someembodiments, the polypeptide inhibits tumor cell growth by 50, 60, 70,80% or more relative to the growth of the tumor in an untreated animal.In some embodiments, the inhibition of tumor cell growth is measured atleast 7 days or at least 14 days after the animals have startedtreatment with the polypeptide. In some embodiments, anotherantineoplastic agent is administered to the animal with the polypeptide.

In certain aspects, the disclosure provides methods for treating asubject having a condition which responds to the inhibition of EGFR,i.e., an “EGFR-associated disease”. Such a method may compriseadministering to said subject an effective amount of any of the EGFRinhibiting polypeptides described herein. In some embodiments, the EGFRbinding polypeptide inhibits EGFR signaling. In some embodiments, theEGFR binding polypeptide inhibits EGFR binding to one or more EGFRligands.

The term “EGFR-associated disease” relates to pathological states whichare dependent on EGFR activity. EGFR is involved either directly orindirectly in the signal transduction pathways of various cellactivities, including proliferation, adhesion and migration, as well asdifferentiation. The diseases associated with EGFR activity include theproliferation of tumour cells, pathological neovascularisation, whichpromotes the growth of solid tumours, neovascularisation in the eye(diabetic retinopathy, age-induced macular degeneration and the like)and inflammation (psoriasis, rheumatoid arthritis and the like).

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptides for the treatment and/or prophylaxis oftumours and/or tumour metastases, where the tumour is particularlypreferably selected from the group consisting of brain tumour, tumour ofthe urogenital tract, tumour of the lymphatic system, stomach tumour,laryngeal tumour, monocytic leukaemia, lung adenocarcinoma, small-celllung carcinoma, pancreatic cancer, glioblastoma and breast carcinoma,without being restricted thereto.

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptide for the treatment of diseases selected from thegroup of cancerous diseases consisting of squamous cell carcinoma,bladder cancer, stomach cancer, liver cancer, kidney cancer, colorectalcancer, breast cancer, head cancer, neck cancer, oesophageal cancer,gynecological cancer, thyroid cancer, lymphoma, chronic leukaemia andacute leukaemia.

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptide t for the treatment and/or prophylaxis ofdiseases caused, mediated and/or propagated by angiogenesis. A diseaseof this type involving angiogenesis is an ocular disease, such asretinal vascularisation, diabetic retinopathy, age-induced maculardegeneration and the like.

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptide for the treatment and/or prophylaxis ofdiseases selected from the group consisting of retinal vascularisation,diabetic retinopathy, age-induced macular degeneration and/orinflammatory diseases.

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptide for the treatment and/or prophylaxis ofdiseases selected from the group consisting of psoriasis, rheumatoidarthritis, contact dermatitis, delayed hypersensitivity reaction,inflammation, endometriosis, scarring, benign prostate hyperplasia,immunological diseases, autoimmune diseases and immunodeficiencydiseases.

In certain aspects, the disclosure provides methods for administeringEGFR binding polypeptide for the treatment and/or prophylaxis of bonepathologies selected from the group consisting of osteosarcoma,osteoarthritis and rickets.

One aspect of the application provides EGFR binding polypeptides thatinhibit EGFR tyrosine phosphorylation or receptor levels in vivo orboth. In one embodiment, administration of an EGFR binder to an animalcauses a reduction in EGFR phosphotyrosine signal in EGFR-expressingtumors. In some embodiments, the EGFR binder causes a reduction inphosphotyrosine signal by at least 20%. In some embodiments, the EGFRbinder causes a decrease in phosphotyrosine signal by at least 50, 60,70, 80, 90% or more.

Additional Agents that May be Used with Appropriate Embodiments of theInvention

One aspect of the invention provides EGFR binding polypeptides linked toa cytotoxic agent. Such embodiments can be prepared by in vitro or invivo methods as appropriate. In vitro methods, include conjugationchemistry well know in the art including chemistry compatible withproteins, such as chemistry for specific amino acids, such as Cys andLys. In order to link a cytotoxic agent to a polypeptide, a linkinggroup or reactive group is used. Suitable linking groups are well knownin the art and include disulfide groups, thioether groups, acid labilegroups, photolabile groups, peptidase labile groups and esterase labilegroups. Preferred linking groups are disulfide groups and thioethergroups. For example, conjugates can be constructed using a disulfideexchange reaction or by forming a thioether bond between the antibodyand the cytotoxic agent. Preferred cytotoxic agents are maytansinoids,taxanes and analogs of CC-1065.

In some embodiments, an EGFR binding polypeptide is linked to abacterial toxin, a plant toxin, ricin, abrin, a ribonuclease (RNase),DNase I, a protease, Staphylococcal enterotoxin-A, pokeweed antiviralprotein, gelonin, diphtherin toxin, Pseudomonas exotoxin, Pseudomonasendotoxin, Ranpimase (Rap), Rap (N69Q), an enzyme, or a fluorescentprotein.

In some embodiments, an EGFR binding polypeptide is linked tomaytansinoids or maytansinoid analogs. Examples of suitablemaytansinoids include maytansinol and maytansinol analogs. Suitablemaytansinoids are disclosed in U.S. Pat. Nos. 4,424,219; 4,256,746;4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650;4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 6,333,410;5,475,092; 5,585,499; and 5,846,545.

In some embodiments, an EGFR binding polypeptide is linked to a taxanes.Taxanes suitable for use in the present invention are disclosed in U.S.Pat. Nos. 6,372,738 and 6,340,701.

In some embodiments, an EGFR binding polypeptide is linked to CC-1065 orits analogs. CC-1065 and its analogs are disclosed in U.S. Pat. Nos.6,372,738; 6,340,701; 5,846,545 and 5,585,499.

An attractive candidate for the preparation of such cytotoxic conjugatesis CC-1065, which is a potent anti-tumor antibiotic isolated from theculture broth of Streptomyces zelensis. CC-1065 is about 1000-fold morepotent in vitro than are commonly used anti-cancer drugs, such asdoxorubicin, methotrexate and vincristine (B. K. Bhuyan et al., CancerRes., 42, 3532-3537 (1982)).

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, andcalicheamicin are also suitable for the preparation of conjugates of thepresent invention, and the drug molecules can also be linked to EGFRbinding polypeptides through an intermediary carrier molecule such asserum albumin.

In other therapeutic treatments or compositions, EGFR bindingpolypeptides are co-administered, or administered sequentially, with oneor more additional therapeutic agents. Suitable therapeutic agentsinclude, but are not limited to, targeted therapeutics, other targetedbiologics, and cytotoxic or cytostatic agents. In some instances in willbe preferred to administer agents from the same or separatetherapeutically acceptable vial, syringe or other administration devicethat holds a liquid formulation.

Cancer therapeutic agents are those agents that seek to kill or limitthe growth of cancer cells while having minimal effects on the patient.Thus, such agents may exploit any difference in cancer cell properties(e.g., metabolism, vascularization or cell-surface antigen presentation)from healthy host cells. Differences in tumor morphology are potentialsites for intervention: for example, the second therapeutic can be anantibody such as an anti-VEGF antibody that is useful in retarding thevascularization of the interior of a solid tumor, thereby slowing itsgrowth rate. Other therapeutic agents include, but are not limited to,adjuncts such as granisetron HCl, androgen inhibitors such as leuprolideacetate, antibiotics such as doxorubicin, antiestrogens such astamoxifen, antimetabolites such as interferon alpha-2a, cytotoxic agentssuch as taxol, enzyme inhibitors such as ras farnesyl-transferaseinhibitor, immunomodulators such as aldesleukin, and nitrogen mustardderivatives such as melphalan HCl, and the like.

The therapeutic agents that can be combined with EGFR bindingpolypeptides for improved anti-cancer efficacy include diverse agentsused in oncology practice (Reference: Cancer, Principles & Practice ofOncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6th edition,Lippincott-Raven, Philadelphia, 2001), such as docetaxel, paclitaxel,doxorubicin, epirubicin, cyclophosphamide, trastuzumab, capecitabine,tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane,goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide,bevacizumab, 5-fluorouracil, leucovorin, levamisole, irinotecan,etoposide, topotecan, gemcitabine, vinorelbine, estramustine,mitoxantrone, abarelix, zoledronate, streptozocin, rituximab,idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine,ibritumomab, tositumomab, interferon alpha-2b, melphalam, bortezomib,altretamine, asparaginase, gefitinib, erlonitib, anti-EGF receptorantibody (e.g., cetuximab or panitumab), ixabepilone, epothilones orderivatives thereof, and conjugates of cytotoxic drugs and antibodiesagainst cell-surface receptors. Preferred therapeutic agents areplatinum agents (such as carboplatin, oxaliplatin, cisplatin), taxanes(such as paclitaxel, docetaxel), gemcitabine, and camptothecin.

The one or more additional therapeutic agents can be administeredbefore, concurrently, or after the EGFR binding polypeptides. Theskilled artisan will understand that for each therapeutic agent theremay be advantages to a particular order of administration. Similarly,the skilled artisan will understand that for each therapeutic agent, thelength of time between which the agent, and an antibody, antibodyfragment or conjugate of the invention is administered, will vary.

Formulation and Administration

Therapeutic formulations comprising EGFR binding polypeptides areprepared for storage by mixing the described proteins having the desireddegree of purity with optional physiologically acceptable carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980)), in the form of aqueous solutions,lyophilized or other dried formulations. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulations herein may also contain more than one active compoundas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. Examples of combinations of active compounds are provided inherein. Such molecules are suitably present in combination in amountsthat are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the proteins of the invention, whichmatrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated proteins of the invention may remain in thebody for a long time, they may denature or aggregate as a result ofexposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

While the skilled artisan will understand that the dosage of eachtherapeutic agent will be dependent on the identity of the agent, thepreferred dosages can range from about 10 mg/square meter to about 2000mg/square meter, more preferably from about 50 mg/square meter to about1000 mg/square meter.

For therapeutic applications, the EGFR binding polypeptides areadministered to a subject, in a pharmaceutically acceptable dosage form.They can be administered intravenously as a bolus or by continuousinfusion over a period of time, by intramuscular, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes. The protein may also be administered by intratumoral,peritumoral, intralesional, or perilesional routes, to exert local aswell as systemic therapeutic effects. Suitable pharmaceuticallyacceptable carriers, diluents, and excipients are well known and can bedetermined by those of skill in the art as the clinical situationwarrants. Examples of suitable carriers, diluents and/or excipientsinclude: (1) Dulbecco's phosphate buffered saline, pH about 7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The method of thepresent invention can be practiced in vitro, in vivo, or ex vivo.

Administration of EGFR binding polypeptides, and one or more additionaltherapeutic agents, whether co-administered or administeredsequentially, may occur as described above for therapeutic applications.Suitable pharmaceutically acceptable carriers, diluents, and excipientsfor co-administration will be understood by the skilled artisan todepend on the identity of the particular therapeutic agent beingco-administered.

When present in an aqueous dosage form, rather than being lyophilized,the protein typically will be formulated at a concentration of about 0.1mg/ml to 100 mg/ml, although wide variation outside of these ranges ispermitted. For the treatment of disease, the appropriate dosage of EGFRbinding polypeptides will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether theantibodies are administered for preventive or therapeutic purposes, thecourse of previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theprotein is suitably administered to the patient at one time or over aseries of treatments.

The present invention also includes kits comprising one or more of theelements described herein, and instructions for the use of thoseelements. In a preferred embodiment, a kit of the present inventionincludes an EGFR binding polypeptides and a therapeutic agent. Theinstructions for this preferred embodiment include instructions forinhibiting the growth of a cancer cell using the EGFR bindingpolypeptide and the therapeutic agent, and/or instructions for a methodof treating a patient having a cancer using the EGFR binding polypeptideand the therapeutic agent.

Preferably, the therapeutic agent used in the kit is selected from thegroup consisting of docetaxel, paclitaxel, doxorubicin, epirubicin,cyclophosphamide, trastuzumab, capecitabine, tamoxifen, toremifene,letrozole, anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin,carboplatin, cisplatin, dexamethasone, antide, bevacizumab,5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide,topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone,abarelix, zoledronate, streptozocin, rituximab, idarubicin, busulfan,chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab,tositumomab, interferon alpha-2b, melphalam, bortezomib, altretamine,asparaginase, gefitinib, erlonitib, anti-EGF receptor antibody (e.g.,cetuximab or panitumumab), ixabepilone, and an epothilone or derivativethereof. More preferably, the therapeutic agent is a platinum agent(such as carboplatin, oxaliplatin, cisplatin), a taxane (such aspaclitaxel, docetaxel), gemcitabine, or camptothecin.

The elements of the kits of the present invention are in a suitable formfor a kit, such as a solution or lyophilized powder. The concentrationor amount of the elements of the kits will be understood by the skilledartisan to varying depending on the identity and intended use of eachelement of the kit.

The cancers and cells there from referred to in the instructions of thekits include breast cancer, colon cancer, ovarian carcinoma,osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovialcarcinoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma,and rhabdomyosarcoma.

The dosage of cytotoxic or therapeutic agents administered in themethods described herein can be readily determined by those skilled inthe art. Pharmaceutical package inserts may also be consulted whendetermining the proper dosage.

Additional Patent References

Methods and compositions described in the following additional patentapplications and patents are also included in this disclosure:

U.S. Publication Nos. 20050186203; 20050084906; 20050008642;20040202655; 20040132028; 20030211078; 20060083683; 20060099205;20060228355; 20040081648; 20040081647; 20050074865; 20040259155;20050038229; 20050255548; 20060246059; and U.S. Pat. Nos. 5,707,632;6,818,418; and 7,115,396; and PCT International Application PublicationNos. WO2005/085430; WO2004/019878; WO2004/029224; WO2005/056764;WO2001/064942; and WO2002/032925.

INCORPORATION BY REFERENCE

All documents and references, including patent documents and websites,described herein are individually incorporated by reference to into thisdocument to the same extent as if there were written in this document infull or in part.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention. While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madethereto without departing from the spirit and scope thereof.

Example 1 Initial Identification of EGFR Binding Molecules

A library of approximately 10¹³ RNA-protein fusion variants wasconstructed based on the scaffold of the tenth type 3 domain of humanfibronectin with three randomized regions at positions 23-29, 52-55 and77-86, amino acid numbering according to SEQ ID NO: 1 (the “NNS”library) (Xu et al, Chemistry & Biology 9:933-942, 2002). Similarlibraries were constructed in which mixtures of phosphoramidite trimerswere used instead of degenerate codons to give randomized regionslacking tryptophan, phenylalanine and cysteine (the “—WFC” library), orlacking tryptophan, phenylalanine, cysteine, leucine, isoleucine,methionine and valine (the “NVH” library). After conversion to mRNA/cDNAheteroduplexes, libraries of a trillion or more mRNA/cDNA-proteinfusions each were incubated with 100 nM EGFR-Fc in solution, andexisting complexes were captured on protein G-coated magnetic beads. ThecDNA was eluted by treatment with high pH, amplified by PCR, and wereused to generate new more focused libraries of mRNA/cDNA-protein fusionsthat were enriched for binders of EGFR. Five cycles of amplification andselection were carried out in this manner and target binding wasmonitored by quantitative PCR. Analogous experiments were carried outusing the s525 variant of EGFR-Fc (amino acids 1-525 of EGFR fused toFc), or using EGFR-Fc in the presence of EGF. In each case, theRNA-protein fusion library bound to the target after 5 cycles ofselection.

Example 2 Identification of EGFR Binding Clones

Proteins encoded by independent clones were analyzed for binding to thefull length ectodomain of EGFR as well as a truncated version containingthe first 525 amino acids (EGFR 525) of the EGFR ectodomain in singlepoint direct binding assays. Anti-His antibodies were used to captureprotein clones in an oriented fashion followed by incubation with fulllength EGFR or EGFR 525 Fc fusion proteins. Bound receptor-Fcs weredetected via anti-human Fc HRP conjugate with a chromogenic readout(i.e. A450). Representative results (partial) from the screening arepresented in Table 1 where the relative binding strengths of 24 clonesto full length and truncated EGFRs are shown. Compared to the control(SGE), all clones demonstrate significant EGFR binding. The SGE controlis the ¹⁰FN3 domain as shown in SEQ ID NO: 1 with the integrin bindingdomain (RGD) substituted with amino acids SGE.

TABLE 1 EGFR-FL-Fc EGFR 525-Fc Clone A450 A450 SEQ ID NO: SGE 0.14340.1385 679A01 1.568 1.5285 207 679A05 1.428 1.7188 208 679A10 0.7870.9251 209 679B01 0.213 0.5389 210 679B09 0.455 0.3754 211 679B10 1.3761.7437 212 679C10 0.467 0.803 213 679F03 2.367 2.4468 214 679F09 2.4162.4685 215 679G06 2.102 0.1282 216 867A01 1.4045 0.1369 217 867E021.4179 1.4313 218 867A03 2.2985 1.8519 219 867B04 2.8105 2.5532 220867C04 1.9493 1.5665 221 867A05 1.8684 1.6112 222 867B05 2.8208 2.5487223 867E05 1.7157 1.6823 224 867C07 2.4325 2.1811 225 867B08 2.61062.4947 226 867H08 1.8953 1.7903 227 867B09 2.7704 2.4136 228 867B102.955 2.5545 229 867F10 2.7801 2.3242 230

Example 3 Cell-Based Competitive Ligand Binding Assay

The Cell-Based Competitive Ligand Binding Assay measures the ability ofa test sample to bind to EGFR on the surface of human A431 cells andcompete with the binding of the natural EGF ligand labeled with aeuropium tag (Eu-EGF). Competition of the test sample with Eu-EGF forbinding to the EGFR's on the cell surface is measured by a decrease influorescent signal. Competitive binding of an anti-EGFR antibody (LA-1)is compared to the binding of two EGFR binding clones (679F03, 679F09and 867A01) in FIGS. 2-4.

Example 4 Initial Optimization of an EGFR-Specific Clone

In order to identify clones with improved affinity and specificity,further mutagenesis was performed to create more focused libraries tooptimize the loops binding to EGFR.

Three libraries were constructed in which one loop at a time wasreplaced with random sequence. The libraries were constructed at the DNAlevel as described in the Material and Methods section, except thatrandom sequences in two out of three loops were replaced with fixedsequence corresponding to the clone being optimized.

Amplification, synthesis of mRNA-protein fusions, and affinity selectionwere carried out with these three libraries. Binding percentages weremonitored each round and PROfusion™ was continued until each libraryshowed binding above 1%. At this point, the random loops were amplifiedfrom each library and reassembled to make a master library in which all3 loops were optimized (FIG. 5).

The master library containing all 3 optimized loops was taken throughcycles of amplification, synthesis of mRNA-protein fusions, and affinityselection. EGFR-Fc was used at decreasing concentrations to select forthe highest affinity binders. In round 1, the concentration of EGFR-Fcwas 100 nM. In round 2, the concentration of EGFR-Fc was 1 nM. In rounds3 and 4, the concentration of EGFR-Fc was 0.1 nM.

Example 5 Cell Based Competitive Ligand Binding Assay to AssessOptimized Derivative Clones

Selected optimized clones are compared to a pre-optimized clone fortheir ability to bind to EGFs on the surface of human A431 cells andcompete with the binding of the natural EGF ligand labeled with aeuropium tag (Eu-EGF). Following HTPP and quantitation, many clones mayexhibit superior inhibition to the staring clone with IC₅₀s in the lownM range.

Example 6 Examination of the Thermal Stability of Optimized EGFRCompetitive IC₅₀Clones

One liter E. coli growths of selected EGFR optimized clones are preparedand the proteins are purified. Differential Scanning calorimetry (DSC)is performed to characterize the energetics of unfolding, or meltingtemperature, T_(m), of individual clones.

Example 7 Examination of the Solution Properties of Optimized EGFRCompetitive Clones

One liter E. coli growths of selected optimized clones are prepared andthe proteins are purified. Size-exclusion chromatography (SEC) is usedto determine monomeric behavior. Monomericity is confirmed using SECcombined with Multi-Angle Laser Light Scattering (MALLS). “Classical”light scattering (also known as “static” or “Rayleigh” scattering orMALLS) provides a direct measure of molecular mass. It is therefore veryuseful for determining whether the native state of a protein is amonomer or a higher oligomer, and for measuring the masses of aggregatesor other non-native species.

Example 8 Determination of Binding Affinity and Kinetics of OptimizedEGFR Competitive Clones

One liter E. coli growths of selected optimized clones are prepared andthe proteins are purified. Surface plasmon resonance (BIAcore) analysisis performed using recombinant, immobilized EGFR and solution-phaseclones in order to determine binding kinetics and binding affinities.

Example 9 Determination of Binding of Optimized EGFR Competitive Clonesto Other HER-family Members

One liter E. coli growths of selected optimized clones are prepared andthe proteins are purified. To ensure that the EGFR competitive clonesare indeed specific for EGFR, they are evaluated at high concentrationagainst other HER family members or another unrelated receptor usingBIAcore methodology. The HER family members or another unrelatedreceptor are immobilized onto a BIAcore chip as is an irrelevant proteinto control for non-specific binding. Clones are passed over the chip at10 μM to look for non-specific binding or specificity.

Example 10 Activity of EGFR Clones in Cell-Based Assays

The purified EGFR clones are evaluated in cell-based assays in order toconfirm activity. In this example, results from clone 679F09 (SEQ ID NO:215) are described. Clone 679F09 was screened for the ability todirectly interfere with ligand-stimulated EGFR activation and downstreamMAP kinase signaling. Immunocytochemical assays (In Cell Westerns) wereused to measure 1) total phosphorylation of the EGFR, 2) phosphorylationon tyrosine 1068 of the EGFR, a functionally important residueresponsible for binding of cytosolic signaling proteins and 3) ERKphosphorylation, a component of the MAP kinase pathway that stimulatesgrowth in response to EGFR activation. These assays were carried out inboth A431 epidermoid carcinoma cells and FaDu head & neck carcinomacells.

In FaDu cells, clone 679F09 blocked EGF-stimulated EGFR phosphorylationon tyrosine 1068 with an IC₅₀ of 1.32 μM and blocked EGF-stimulated ERKphosphorylation with an IC₅₀ of 2.8 μM. In A431 cells, clone 679F09blocked EGF-stimulated total EGFR phosphorylation with an IC₅₀ of 2.4μM, EGF-stimulated EGFR phosphorylation on tyrosine 1068 with an IC₅₀ of2.88 μM and blocked EGF-stimulated ERK phosphorylation with an IC₅₀ of3.1 μM.

ELISA assays to measure phosphorylation of AKT, total EGFRphosphorylation, EGFR phosphorylation on tyrosine 1068, and ERKphosphorylation were used to evaluate these same endpoints in DiFi coloncarcinoma cells, as this line was not suitable for In Cell Westernassays. In DiFi cells, clone 679F09 blocked EGF-stimulated AKTphosphorylation with an IC₅₀ between 1.8 μM and 5.3 μM, blockedEGF-stimulated total EGFR phosphorylation with an IC₅₀ of 5.3 μM,blocked EGF-stimulated EGFR phosphorylation on tyrosine 1068 with anIC₅₀ of 1.49 μM, and blocked EGF-stimulated ERK phosphorylation with anIC₅₀ of greater than 4.4 μM.

Example 11 Epitope Mapping

An In Cell Western (ICW) assay was used to determine if EGFR clonescould interfere with the binding of other EGFR antibodies to a knownepitope on the EGFR extracellular domain. A panel of antibodies wasassembled with defined binding regions (Table 2). EGFR clones wereincubated with A431 cells, unbound protein was washed away and boundprotein crosslinked to the receptor with BS3. Cells were fixed andprobed with an antibody where the binding site is known. If the EGFRclone shares common epitopes with the antibody, binding of the clone toEGFR prevents or reduces binding by the antibody.

Table 2 depicts the results of the competition binding studies with EGFRbinding clone 679F09, as well as anti-EGFR antibodies cetuximab andpanitumumab.

TABLE 2 Antibodies to the EGFR extracellular domain with defined bindingdeterminants. Clone SUPPLIER SPECIES EPITOPE Competing 1 Abcam ab38165Rab Peptide AA 42-58 2 E234 Abcam ab32198 Rab Peptide AA 40-80 (No ICC)3 N-20 Santa Cruz#31155 Goat IgG AA 110-160 4 ICR10 Abcam ab231 RatIgG2a AA 124-176^(b), Santa Cruz #57095 neutralizing^(e) 5 EGFR1 Abcamab30 Mu IgG1 AA 176-294, Chemicon neutralizing^(b) MAB88910ab30&MAB88910@ Labvision MS-311 (1 mg/ml) 6 199.12 Labvision MS-396-P MuIgG2a AA 124-176, non- neutralizing^(b) 7 LA22 Upstate 05-104 Mu IgG2aAA 351-364, C, P neutralizing^(a) 8 Abcam ab15669 Rab PeptideAA376-394^(d) 9 225 Sigma E2156 Mu IgG1 AA 294-475, C, P, LabvisionMS-269-P neutralizing^(b,c) 679F09 10 528 Abcam ab3103 Mu IgG2a AA294-475, C, P Santa Cruz#120 neutralizing^(b,c) Labvision MS-268-P 11B1D8 Labvision MS-666-P Mu IgG2a AA 294-475^(b) C, P, 679F09 12 LA1Upstate 05-101 Mu IgG1 neutralizing C, P, 679F09 13 HI1 LabvisionMS-316-P Mu IgG1 AA 294-475, non- 679F09 neutralizing^(b) 14 H1.6Labvision MS-378-P Mu IgG1 AA 294-475, 679F09 neutralizing^(b) 15 29.1Sigma E2760 Mu IgG1 External Abcam ab10414 carbohydrate non-neutralizing Dilute Abs 1:100, weak binders can be diluted 1:50.^(a)JBC264(1989)17469 Ala351-Asp364, ^(b)J Immunological Methods287(2004)147, ^(c)Mol Biol Med1(1983)511, ^(d)Raised against a peptideto mouse EGFR, ^(e)Int J Oncol4(1994)277. C—cetuximab; P—panitumumab.

Example 12 Re-Optimization of Clones

One of the commonly seen degradation processes for proteins duringstorage is the oxidation of methionine or other amino acid residues,such as tryptophan, tyrosine, or histidine. Optimization may beperformed in order to select for clones which retain the desirableproperties of biological activity and biophysical properties of thestarting clone, but are substituted in undesirable methionineresidue(s), if present, in the loops. The same approach is used when anytryptophan, tyrosine, histidine residues in the loops are identified asbeing prone to oxidative damage.

Example 13 Direct Binding Assay for Re-Optimized Derivatives

A direct binding assay is used to screen re-optimized derivatives forenhanced binding to EGFR. Clones that show binding in a single-pointassay are analyzed further using a gradient of clone concentrations.

Example 14 Examination of the Solution Properties of Re-Optimized EGFRCompetitive Clones

Size-exclusion chromatography (SEC) of HTPP material for re-optimizedEGFR competitive clones typically reveals monomeric behavior indicatingthat at higher protein concentration, optimized clones do not have atendency to aggregate.

Example 15 Determination of Binding Affinity and Kinetics ofRe-Optimized EGFR Competitive Clones

One liter E. coli growths of selected FG loop optimized EGFR competitiveclones are prepared and the proteins are purified. BIAcore analysis isperformed using recombinant, immobilized EGFR and solution-phase clonesin order to determine binding kinetics and binding affinities.Typically, binding affinities may range from double-digit to tripledigit pM for the clones.

Example 16 Determination of Binding of Re-Optimized EGFR CompetitiveClones to Other HER family Members

One liter E. coli growths of selected FG loop optimized EGFR competitiveclones is prepared and the proteins are purified. To ensure that theEGFR competitive clones are indeed specific for EGFR, they are evaluatedat high concentration against other HER receptors or another unrelatedreceptor using Biacore methodology. Typically, the majority of cloneswill exhibit no detectable binding to other EGFR family members or otherunrelated receptors at this high concentration illustrating that theseclones are indeed specific to EGFR.

Example 17 Examination of the Thermal Stability of Re-Optimized Clonesto EGFR

One liter E. coli growths of selected re-optimized EGFR competitiveclones are prepared and the proteins are purified. Differential Scanningcalorimetry (DSC) is performed to characterize the energetics ofunfolding, or melting temperature, T_(m), of individual clones.

Example 18 PEGylation of Fn3 Domains

PEGylation of Fn3 domains is performed to enhance pharmacokineticproperties. Optimized clones are produced in an E. coli expressionsystem with a C-terminal cysteine substitution.

SEQ ID NO: 235 is linked to the N-terminus of a clone lacking cysteineresidues. The single sulfhydryl of the cysteine residue from SEQ ID NO:235 is used to couple to PEG variants using standard maleimide chemistryto yield two different PEGylated forms. Both a linear 20 kDabifunctional PEG and a mono-functional branched 40 kDa PEG (NOFCorporation) are conjugated to clones. The PEGylated protein forms arepurified from unreacted protein and PEG by ion exchange andsize-exclusion chromatography. Covalent linkage of the two PEG forms areverified by SDS-PAGE, mass spectrometry and analytical size exclusionchromatography coupled with multi-angle laser light scattering.

Example 19 Determination of Binding Affinity and Kinetics of PEGylatedFn3 Domain Clone Variants

Surface plasmon resonance (Biacore) analysis is performed usingrecombinant, EGFR-Fc captured on anti-human antibody surface andsolution-phase analyte (PEGylated clone) in order to determine bindingkinetics and binding affinities of PEGylated variants.

Example 20 Evaluation of EGFR Binding Clones for AntiproliferativeActivity in an EGFR-Dependent Cell Line

Clones were evaluated for antiproliferative activity in the DiFi coloncarcinoma cell line, which depends on EGFR signaling for growth. Aprimary screen was carried out at 10 μM and 1 μM in duplicate toidentify active clones. An active clone demonstrated greater than 50%inhibition at either concentration and preferably exhibited a doseresponse. Active clones were followed up with IC₅₀ determinations bytesting serial dilutions at eight concentrations in triplicate. The mainassay method measured incorporation of ³H-thymidine into newlysynthesized DNA, but a metabolic detection assay was occasionally usedas well that measured conversion of a water soluble tetrazolium salt toa colored byproduct. Standard compounds were included in each experimentto verify assay performance and reproducibility.

Using the ³H-thymidine incorporation assay in DiFi cells, clone 679F09mildly inhibited proliferation in one experiment, yet failed to inhibitproliferation in two separate experiments.

Example 21 Generation of EGFR/IGF-IR PEGylated Bispecific Molecules

Bispecific molecules directed to both EGFR and IGF-IR are created bylinking together fibronectin based scaffold domain clones specific toeach target using a bifunctional PEG molecule. A cysteine residue issubstituted at the C-terminus of each clone so that the bifunctional PEGcan link the two clones through maleimide chemistry.

In the present example, the IGF-IR binding clone represented in SEQ IDNO: 203 and the EGFR binding clone represented in SEQ ID NO: 231 areutilized. One of two approaches is used to generate the appropriatebispecific molecule. Equimolar mixtures of the EGFR clone, the IGF-IRclone, and maleimide-PEG-X-kDa-maleimide are mixed for the appropriateperiod of time and the products are separated by ion-exchangechromatography under pH conditions optimum for the separation of the twoisolated clones based upon differences in pI. The theoretical productsand their ratios from this separation are 1 part homospecific EGFR, 1part homospecific IGF-IR, and 2 parts bispecific EGFR/IGF-IR. A secondapproach is predicated upon the sequential addition of species toPEG-linker. An excess of linker, for example a 10-fold excess, is addedto the one of the species and the reaction allowed to proceed tocompletion. The PEGylated mono-species is recovered from PEG-linkedhomodimer and unreacted PEG-linker by ion-exchange chromatography. Theisolated PEGylated mono-species is then reacted with an equimolarquantity of the other clone species to create the bispecific molecule.

Example 22 Generation of EGFR/VEGFR2 PEGylated Bispecific Molecules

Bispecific molecules directed to both EGFR and VEGFR2 are created bylinking together fibronectin based scaffold domain clones specific toeach target using a bifunctional PEG molecule. A cysteine residue issubstituted at the C-terminus of each clone so that the bifunctional PEGcan link the two clones through maleimide chemistry.

In the present example, the VEGFR2 binding clone represented in SEQ IDNO: 128 and the EGFR binding clone represented in SEQ ID NO: 231 areutilized. One of two approaches is used to generate the appropriatebispecific molecule. Equimolar mixtures of the EGFR clone, the VEGFR2clone, and maleimide-PEG-X-kDa-maleimide are mixed for the appropriateperiod of time and the products are separated by ion-exchangechromatography under pH conditions optimum for the separation of the twoisolated clones based upon differences in pI. The theoretical productsand their ratios from this separation are 1 part homospecific EGFR, 1part homospecific VEGFR2, and 2 parts bispecific EGFR/VEGFR2. A secondapproach is predicated upon the sequential addition of species toPEG-linker. An excess of linker, for example a 10-fold excess, is addedto the one of the species and the reaction allowed to proceed tocompletion. The PEGylated mono-species is recovered from PEG-linkedhomodimer and unreacted PEG-linker by ion-exchange chromatography. Theisolated PEGylated mono-species is then reacted with an equimolarquantity of the other clone species to create the bispecific molecule.

Example 23 Generation of EGFR/EGFR PEGylated Bispecific Molecules

Bidomain molecules directed to EGFR are created by linking togetherfibronectin based scaffold domain clones specific to EGFR using abifunctional PEG molecule. A cysteine residue is substituted at theC-terminus of each clone so that the bifunctional PEG can link the twoclones through maleimide chemistry.

In the present example, the EGFR binding clone represented in SEQ ID NO:231 is utilized. A 2:1 ration of the EGFR clone andmaleimide-PEG-X-kDa-maleimide are mixed for the appropriate period oftime and the products are separated by ion-exchange chromatography. Thetheoretical products and their ratios from this separation are 2 partshomospecific EGFR and 2 parts EGFR/EGFR.

Example 24 Generation of EGFR/IGF-IR Polypeptide-Linked BispecificMolecules

Bispecific molecules directed to both EGFR and IGF-IR are created byconnecting the two clones via a polypeptide linker. The orientation ofthe two clones with regards to the N-terminal clone versus theC-terminal clone is arbitrary, but in the present example, the EGFRclone is in the N-terminal position. In the present example, the EGFRbinding clone represented in SEQ ID NO: 215 and the IGF-IR binding clonerepresented in SEQ ID NO: 236 are utilized.

Hence the DNA construct in the present example is EGFR bindingclone-polypeptide linker-IGFR binding clone. This is expressed in E.coli and purified per usual either from inclusion bodies or from thesoluble fraction.

Example 25 Generation of EGFR/VEGFR2 Polypeptide-Linked BispecificMolecules

Bispecific molecules directed to both EGFR and VEGFR2 are created byconnecting the two clones via a polypeptide linker. The orientation ofthe two clones with regards to the N-terminal clone versus theC-terminal clone is arbitrary, but in the present example, the EGFRclone is in the N-terminal position. In the present example, the EGFRbinding clone represented in SEQ ID NO: 215 and the VEGFR2 binding clonerepresented in SEQ ID NO: 129 are utilized.

Hence the DNA construct in the present example is EGFR bindingclone-polypeptide linker-VEGFR binding clone. This is expressed in E.coli and purified per usual either from inclusion bodies or from thesoluble fraction.

Example 28 Generation of EGFR/EGFR Polypeptide-Linked BispecificMolecules

Bisdomain molecules directed to EGFR are created by connecting the twoclones via a polypeptide linker. In the present example, the EGFRbinding clone represented in SEQ ID NO: 215 is utilized. Hence the DNAconstruct in the present example is EGFR binding clone-polypeptidelinker-EGFR binding clone. This is expressed in E. coli and purified perusual either from inclusion bodies or from the soluble fraction.

Material and Methods Used Herein

The following materials and methods were used for the experimentsdescribed in the Examples.

Recombinant Proteins:

EGFR-Fc (R&D Systems, Minneapolis, Minn.) consisting of the ectodomainof human EGFR as a Fc fusion was purchased and shown to be functionalfor EGF binding by Biacore. The first 525 amino acids of the human EGFRextracellular domain was cloned into a mammalian expression vectorcontaining the hinge and constant regions of human IgG1. Transienttransfection of the plasmid produced a fusion protein, EGFR 525-Fc,which was subsequently purified by Protein A chromatography. Thisprotein was shown to be capable of binding EGF using a similar Biacoreassay as was done for the full length ectodomain fusion.

Primers:

The following oligonucleotides were prepared by chemical synthesis foreventual use in library construction and mutagenesis of selected clones.

T7TMV: 5′-TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT ACA ATG-3′(SEQ ID NO: 237) FnAB: 5′-GGG ACA ATT ACT ATT TAC AAT TAC AAT GGT TTCTGA TGT GCC GCG CGA CCT GGA AGT GGT TGC TGC CAC CCC CAC CAG CCT GCT GATCAG CTG G-3′ (SEQ ID NO: 238) FnBC: 5′-AGC CTG CTG ATC AGC TGG NNS NNSNNS NNS NNS NNS NNS CGA TAT TAC CGC ATC ACT-3′ (SEQ ID NO: 239) FnBC8:5′-AGC CTG CTG ATC AGC TGG NVH NVHNVH NVH NVH NVH NVH NVH TAT TAC CGCATC ACT-3′ (SEQ ID NO: 240)

FnBC (trimer): 5′-AGC CTG CTG ATC AGC TGG X X X X X X X CGA TAT TAC CGCATC ACT-3′ (SEQ ID NO: 241) to construct a BC loop using trimerphosphoramidites

FnCD: 5′-AGG CAC AGT GAA CTC CTG GAC AGG GCT ATT GCC TCC TGT TTC GCC GTAAGT GAT GCG GTA ATA TCG-3′ (SEQ ID NO: 242) FnDE: 5′-CAG GAG TTC ACT GTGCCT NNS NNS NNS NNS ACA GCT ACC ATC AGC GGC-3′ (SEQ ID NO: 243)

FnDE (trimer): 5′-CAG GAG TTC ACT GTG CCT X X X X ACA GCT ACC ATC AGCGGC-3′ (SEQ ID NO: 244) to construct a DE loop using trimerphosphoramidites

FnEF: 5′-AGT GAC AGC ATA CAC AGT GAT GGT ATA ATC AAC GCC AGG TTT AAG GCCGCT GAT GGT AGC TGT-3′ (SEQ ID NO: 245)

FnFG6: 5′-ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNS CCA ATT TCCATT AAT TAC-3′ (SEQ ID NO: 246) to give an FG loop with 6 random aminoacids.FnFG6 (trimer): 5′-ACT GTG TAT GCT GTC ACT X X XXX X CCA ATT TCC ATT AATTAC-3′ (SEQ ID NO: 247) to give an FG loop with 6 random amino acidsusing trimer phosphoramidites.FnFG8: 5′-ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNS NNS NNS CCAATT TCC ATT AAT TAC-3′ (SEQ ID NO: 248) to give an FG loop with 8 randomamino acids.FnFG8 (trimer): 5′-ACT GTG TAT GCT GTC ACT XX X X XXX X CCA ATT TCC ATTAAT TAC-3′ (SEQ ID NO: 249) to give an FG loop with 8 random amino acidsusing trimer phosphoramiditesFnFG10: 5′-ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNS NNS NNS NNSNNS CCA ATT TCC ATT AAT TAC-3′ (SEQ ID NO: 250) to give an FG loop with10 random amino acids.FnFG10: 5′-ACT GTG TAT GCT GTC ACT XX XX XX XXX X CCA ATT TCC ATT AATTAC-3′ (SEQ ID NO: 251) to give an FG loop with 10 random amino acidsusing trimer phosphoramidites.FnFG12: 5′-ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNS NNS NNS NNSNNS NNS NNS CCA ATT TCC ATT AAT TAC-3′ (SEQ ID NO: 252) to give an FGloop with 12 random amino acids.FnFG14: 5′-ACT GTG TAT GCT GTC ACT NNS NNS NNS NNS NNS NNS NNS NNS NNSNNS NNS NNS NNS NNSCCA ATT TCC ATT AAT TAC-3′ (SEQ ID NO: 253) to givean FG loop with 14 random amino acids.

FnG: 5′-TTA AAT AGC GGA TGC CTT GTC GTC GTC GTC CTT GTA GTC TGT GCG GTAATT AAT GGA AAT TGG-3′ (SEQ ID NO: 254) FLAG: 5′-TTT TTT TTT TTT TTT TTTTTA AAT AGC GGA TGC CTT GTC GTC GTC GTC CTT GTA-3′ (SEQ ID NO: 255)679F09BC: 5′-AGC CTG CTG ATC AGC TGG CAG GTT CCG CGT CCG ATG TAC CAA TATTAC CGC ATC ACT TAC-3′ (SEQ ID NO: 256) 679F09DE: 5′-CAG GAG TTC ACT GTGCCT GGT GGT GTT CGT ACA GCT ACC ATC AGC GGC-3′ (SEQ ID NO: 257)679F09FG: 5′-ACT GTG TAT GCT GTC ACT GAC TAC ATG CAT TCT GAA TAC CGT CAGTAC CCA ATT TCC ATT AAT TAC-3′ (SEQ ID NO: 258) FnCD′: 5′-AGG CAC AGTGAA CTC CTG GAC AGG GCT ATT GCC TCC TGT TTC GCC GTA AGT GAT GCG GTAATA-3′ (SEQ ID NO: 259) FnB: 5′-AGC CTG CTG ATC AGC TGG-3′ (SEQ ID NO:260) FnD: 5′-CAG GAG TTC ACT GTG CCT-3′ (SEQ ID NO: 261) FnF: 5′-ACT GTGTAT GCT GTC ACT-3′ (SEQ ID NO: 262) Primary Library Construction:

A diverse library was constructed by extension of the overlappingsynthetic oligonucleotides listed above, using KOD polymerase (EMDBiosciences, San Diego, Calif.). In these oligonucleotide designations,“N” indicates a mixture of A, C, G and T; “V” indicates a mixture of A,C and G, “H” indicates a mixture of A, C and T, and “S” indicates amixture of C and G. Loop definitions are identical to those describedpreviously (Xu et al, 2002). The BC loop was constructed by extension of50 pmol FnBC with 100 pmol FnCD in a 100 μl KOD reaction that wassupplemented with 1 M Betaine and 3% DMSO. The reaction was takenthrough 10 temperature cycles of 30 s at 94° C., 30 s at 52° C. and 1min at 68° C. to ensure complete extension of the fragments. The DE andFG loops were constructed in a similar manner, using 200 pmol FnDE with100 pmol FnEF for the DE loop and 100 pmol FnFG10 with 200 pmol FnG forthe FG loop. Following extension of the 3 individual loops, the DE andFG loops were combined and extended together for an additional 10temperature cycles of 30 s at 94° C., 30 s at 52° C. and 1 min at 68° C.The BC loop was extended with 100 pmol FnAB in the same manner. TheDE/FG mixture and the BC loop were each diluted 10-fold with fresh KODreagents and extended with FLAG and T7TMV respectively, for 10temperature cycles 30 s at 94° C., 30 s at 52° C. and 1 min at 68° C.Finally the fragments were combined and extended together for 10temperature cycles of 30 s at 94° C., 30 s at 52° C. and 1 min at 68° C.This produced a library with 7 random amino acids in the BC loop, 4random amino acids in the DE loop, and 10 random amino acids in the FGloop. Additional libraries were constructed with different FG looplengths by using oligonucleotides FnFG6, FnFG8, FnFG12 or FnFG14 insteadof FnFG10. The libraries containing FG loop lengths between 6 and 14amino acids were combined to give the “NNS” library.

The “NVH” and “WFC” libraries were made using the same method, exceptthat oligonucleotides FnBC, FnDE and FnFG were replaced with FnBC(trimer), FnDE (trimer) and FnFG (trimer) respectively. In theseoligonucleotides, X denotes a mixture of trimer phosphoramidites (GlenResearch, Sterling, Va.) that encode 13 amino acids for the “NVH”library (Lys, Asn, Thr, Gln, His, Pro, Arg, Glu, Asp, Ala, Gly, Tyr,Ser) or 17 amino acids for the “—WFC” library (Lys, Asn, Thr, Gln, His,Pro, Arg, Glu, Asp, Ala, Gly, Tyr, Ser, Leu, Ile, Met, Val).

RNA-Protein Fusion Production:

Double-stranded DNA libraries were converted into RNA-protein fusions(PROfusion™) essentially as described (Xu et al, 2002, Kurz et al, Nuc.Acid Res. 28:83, 2000). Briefly, the DNA library was transcribed usingan in vitro transcription kit (MEGAscript™, Ambion, Austin, Tex.) andthe resulting RNA was desalted by size exclusion chromatography on aNAP-5 column (GE Healthcare). 2 nmol RNA was crosslinked by irradiationat 314 nM for 20 min in 200 μl of a solution containing 150 mM NaCl, 10mM Tris-HCl (pH8) and a 1.5-fold excess of puromycin-containing linker(5′-Pso u agc gga ugc XXX XXX CC Pu-3′ (Pu=puromycin, Pso=C6-psoralen,u, a, g, c=2′-OMe-RNA, X=9-atom PEG spacer.))

The mRNA-puromycin molecules were then translated in 3 ml rabbitreticulocyte lysate (Ambion, Austin, Tex.). The resulting mRNA-proteinfusions were purified using oligo dT cellulose (GE Healthcare) andreverse-transcribed using 2 nmol of the primer FLAG and superscript IIreverse transcriptase (Invitrogen) according to the manufacturersinstructions.

The cDNA/mRNA-protein fusions were purified by M2 Flag agarose (Sigma,St Louis, Mo.) and quantitated by PCR. This gave a purified library ofapproximately 10¹² full length clones that were amplified by PCR usingprimers T7TMV and FLAG for use in subsequent PROfusion™ experiments.

Affinity Selection for Binding to EGFR:

Human IgG (Sigma) was coated on protein C-coated magnetic beads(Invitrogen) to produce a negative selection matrix. The RNA-proteinfusion library was mixed with the negative selection matrix to removeclones that bind to protein G or the Fc region of the target. The beadswere separated on a magnet and the unbound fraction was collected andadded to 100 nM EGFR-Fc in PBS with 0.05% Tween 20 and 1 mg/ml BSA(Ambion). After 30 min, the bound proteins were captured on ProteinG-coated magnetic beads and were washed 6 times using a Kingfishermagnetic particle processor (Thermo Electron, Waltham, Mass.). The cDNAwas eluted in 100 mM KOH, was neutralized with 100 mM Tris-HCl, and wasamplified by PCR to generate a second-generation library enriched inmolecules that bound EGFR. The consecutive processes of amplification,synthesis of RNA-protein fusions, and affinity selection were carriedout a total of 5 times and the bound molecules were quantitated by PCR.The binding populations obtained after rounds 4 and 5 were amplified andligated by recombination (InFusion™, Clontech, Mountain View, Calif.)into an E. coli expression vector containing a promoter for T7 RNApolymerase and an in-frame His₆ tag. The ligated mixture was transformedinto E. coli strain BL21 (DE3) pLysS (Invitrogen) that expresses T7 RNApolymerase upon induction with IPTG, thereby giving inducible proteinexpression.

Construction of Single-Loop Randomized Libraries from Clone 679F09

Three libraries were constructed in which single loops were replacedwith random sequence. The libraries were constructed at the DNA level byoverlap extension with KOD polymerase as described above, except thatrandom sequences in two out of three loops were replaced with fixedsequence corresponding to clone 679F09. The fixed sequences wereprovided in the BC loop by oligonucleotide 679F09BC, and in the DE loopby oligonucleotide 679F09DE, and in the FG loop by oligonucleotide679F09FG. These replaced the corresponding random oligonucleotides FnBC,FnDE, or FnFG10 during library construction. Randomization of positionXX necessitated the use of primer FnCD′ in place of FnCD, and FnBC8 inplace of FnBC. In addition, the use of “NVH” codons removed hydrophobicamino acids from the library.

The library in which the BC loop from clone 679F09 was replaced byrandom amino acids was made by assembling the oligonucleotides:T7TMV+FnAB+FnBC8+FnCD′+679F09DE+FnEF+679F09FG+FnG+FLAG as describedabove. The library in which the DE loop from clone 679F09 was replacedby random amino acids was made by assembling the oligonucleotides:T7TMV+FnAB+679F09BC+FnCD′+FnDE (trimer)+FnEF+679F09FG+FnG+FLAG. Thelibrary in which the FG loop from clone 679F09 was replaced by randomamino acids was assembled from the oligonucleotides:T7TMV+FnAB+669F09BC+FnCD′+679F09+FnEF+FnFG10 (trimer)+FnG+FLAG.

Construction of 3-Loop Randomized Libraries from Clone 679F09

The three single-loop libraries derived from clone 679F09 were subjectedto PROfusion™ selection as described above. The surviving clones in eachlibrary contained 2 loops from the parent clone and 1 loop from therandom sequence that was compatible with binding of each clone. Thethree random loops, one from each library, were amplified usingoligonucleotide primers that bound the surrounding scaffold regions. TheBC loop was amplified from the library containing the variable BC loopusing oligonucleotide primers FnB and FnCD′. The DE loop was amplifiedfrom the library containing the variable DE loop using oligonucleotideprimers FnD and FnEF. The FG loop was amplified from the librarycontaining the variable FG loop using oligonucleotide primers FnF andFnG. The three excised loops were purified by agarose gelelectrophoresis, mixed in equal proportions and amplified by KODpolymerase with primers FnAB and FnG followed by T7TMV and FLAG.

Optimization of Clones Containing 3 Random Loops:

The 3-loop randomized library was taken through cycles of amplification,synthesis of RNA-protein fusions, and affinity selections as describedabove. EGFR-Fc was used at decreasing concentrations to select for thehighest affinity binders. After 4 rounds, the resulting proteinpopulations were cloned into E. coli for analysis as described above.

Direct Binding ELISA for EGFR

The protein populations resulting from the above selections areexpressed in E. coli as His6 tagged proteins. The direct binding ELISArelies on the oriented capture of His6 tagged clones onto anti-Hismonoclonal antibody plates (EMD Biosciences, San Diego, Calif.) blockedwith casein block buffer (Pierce, Rockford, Ill.) for 1-2 hrs. Typicallya 1:50 dilution of HTPP material (0.2-2 ug) is captured for 1 hr,followed by incubation with 50 nM of the EGFR-Fc targets. Bound EGFR-Fcsare detected by incubation with anti-human HRP. Bound HRP conjugate isdetected using the colorimetric substrate TMB (BD Biosciences, San Jose,Calif.) following the manufacturer's instructions.

Cell-Based Competitive Ligand Binding Assay

The clones resulting from the above selections are assayed in theCell-Based Competitive Ligand Binding Assay to determine if the clonesare competitive with EGF, the natural ligand, for binding to the EGFRlocated on the surface of cells. Human epithelial carcinoma cells, cloneA431, which express large numbers of EGFRs on the cell surface, areplated into 96 well tissue culture plates and allowed to adhere for 48hours. The cells are washed with serum free media and dilutions of theselected clones are incubated with the cells for 15 minutes, 37° C. in ahumidified incubator in 5% CO₂. This incubation allows binding of theclone to the EGFR on the cell surface. Europium labeled EGF (Eu-EGF) ata final concentration of 10 nM is then added to the cells and incubatedfor an additional 3 hours at 4° C. After incubation, the cells arewashed with cold PBS to remove unbound clones and Eu-EGF and 50 ul ofEnhancement Solution (Perkin Elmer) is added to the cells and incubatedfor 45 mins at 37° C. This step dissociates the europium label from theEGF ligand producing a fluorescent signal which is measured by timeresolved fluorescence. The intensity of the signal is correlated withthe amount of Eu-EGF bound to the cell's surfaces. A dose responsedecrease in signal intensity indicates that a given clone competes withthe europium labeled natural EGF ligand for binding to the EGFR.

High Throughput Protein Production (HTPP):

Selected binders cloned into pDEST-14 vector and transformed into E.coli BL21 (DE3) pLysS cells are inoculated in 5 ml LB medium containing50 μg/mL carbenicillin and 34 μg/mL chloromphenicol in a 24-well formatand grown at 37° C. overnight. Fresh 5 ml LB medium (50 μg/mLcarbenicillin and 34 μg/mL chloromphenicol) cultures are prepared forinducible expression by aspirating 200 μl from the overnight culture anddispensing it into the appropriate well. The cultures are grown at 37°C. until A₆₀₀ 0.6-1.0. After induction with 1 mMisopropyl-β-thiogalactoside (IPTG) the culture is grown for another 4hours at 30° C. and harvested by centrifugation for 30 minutes at 3220 gat 4° C. Cell Pellets are frozen at −80° C.

Cell pellets (in 24-well format) are lysed by resuspension in 450 μl ofLysis buffer (50 mM NaH₂PO₄, 0.5 M NaCl, 1× Complete™ Protease InhibitorCocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM Imidazole, 1mg/ml lysozyme, 30 ug/ml DNAse, 2 ug/ml aprotonin, pH 8.0) and shaken atroom temperature for 1 hour. Lysates are clarified and re-racked into a96-well format by transfer into a 96-well Whatman GF/D Unifilter fittedwith a 96-well, 650 μl catch plate and are centrifuged for 5 minutes at200 g. The clarified lysates are transferred to a 96-well Ni-ChelatingPlate that has been equilibrated with equilibration buffer (50 mMNaH₂PO₄, 0.5 M NaCl, 10 mM CHAPS, 40 mM Imidazole, pH 8.0) and isincubated for 5 min. Unbound material is removed by vacuum. The resin iswashed 2×0.3 ml/well with Wash buffer #1 (50 mM NaH₂PO₄, 0.5 M NaCl, 5mM CHAPS, 40 mM Imidazole, pH 8.0) with each wash removed by vacuum.Next the resin is washed with 3×0.3 ml/well with PBS with each wash stepremoved by vacuum. Prior to elution each well is washed with 50 μlElution buffer (PBS+20 mM EDTA), incubated for 5 min and this wash isdiscarded by vacuum. Protein is eluted by applying an additional 100 ulof Elution buffer to each well. After a 30 minute incubation at roomtemperature the plate(s) are centrifuged for 5 minutes at 200 g andeluted protein is collected in 96-well catch plates containing 5 μl of0.5M MgCl₂ affixed to the bottom of the Ni-plates. Eluted protein isquantified using a BCA assay with lysozyme as the protein standard.

Midscale Expression and Purification of Soluble Fibronectin-BasedScaffold Protein Binders:

For expression, selected clone(s), followed by the His₆ tag, are clonedinto a pET9d (EMD Biosciences, San Diego, Calif.) vector and areexpressed in E. coli BL21 (DE3) pLysS cells. Twenty ml of an inoculumculture (generated from a single plated colony) is used to inoculate 1liter of LB medium containing 50 μg/mL carbenicillin and 34 μg/mLchloromphenicol. The culture is grown at 37° C. until A₆₀₀ 0.6-1.0.After induction with 1 mM isopropyl-β-thiogalactoside (IPTG) the cultureis grown for 4 hours at 30° C. and is harvested by centrifugation for 30minutes at >10,000 g at 4° C. Cell Pellets are frozen at −80° C. Thecell pellet is resuspended in 25 mL of lysis buffer (20 mM NaH₂PO₄, 0.5M NaCl, 1×Complete™ Protease Inhibitor Cocktail-EDTA free (Roche), 1 mMPMSF, pH 7.4) using an Ultra-turrax homgenizer (IKA works) on ice. Celllysis is achieved by high pressure homongenization (>18,000 psi) using aModel M-110S Microfluidizer (Microfluidics). The soluble fraction isseparated by centrifugation for 30 minutes at 23,300 g at 4° C. Thesupernatant is clarified via 0.45 μm filter. The clarified lysate isloaded onto a HisTrap column (GE) pre-equilibrated with 20 mM NaH₂PO₄,0.5 M NaCl, pH 7.4. The column is then washed with 25 column volumes of20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4, followed by 20 column volumes of 20mM NaH₂PO₄, 0.5 M NaCl, 25 mM imidazole pH 7.4, and then 35 columnvolumes of 20 mM NaH₂PO₄, 0.5 M NaCl, 40 mM imidazole pH 7.4. Protein iseluted with 15 column volumes of column volumes of 20 mM NaH₂PO₄, 0.5 MNaCl, 500 mM imidazole pH 7.4, fractions are pooled based on absorbanceat A₂₈₀ and are dialyzed against 1×PBS, 50 mM Tris, 150 mM NaCl, pH 8.5or 50 mM NaOAc; 150 mM NaCl; pH4.5. Any precipitate is removed byfiltering at 0.22 μm.

PEGylation of EGFR Binding Clones

The EGFR binding clone-PEG40 molecule is prepared by using a 2-foldexcess of PEG-40-kDa (NOF Corporation) to the C-version of the clone viamaleimide chemistry. The reaction is allowed to proceed at roomtemperature for 2.5 hours. Free PEG-40 is separated from clone-PEG-40 byCation Exchange Chromatography (SP-HiTrap; GE). The reaction mixture isdiluted 1:10 with 20 mM NaH₂PO₄, pH 6.7 and is applied to an SP-HiTrapcolumn pre-equilibrated with Equilibration buffer (20 mM NaH₂PO₄, 10 mMNaCl, pH 6.7), washed with Equilbration buffer and is eluted using 20 mMNaH₂PO₄, 0.5M NaCl, pH 6.7. Eluted fractions are pooled based onSDS-PAGE analysis. The SP-pooled eluate is buffer exchanged via G25Chromatography (GE) into PBS.

The PEG20-EGFR binding clone-PEG20 is prepared by using a 2-fold excessof purified clone-C to PEG-20-kDa (NOF Corporation) via maleimidechemistry. The reaction is carried out at room temperature for 1 hour.Unpegylated protein is separated from the PEG20-clone-PEG20 by SECChromatography (Superose 6; GE) in 20 mM NaH₂PO₄, 10 mM NaCl, pH 6.7buffer. The fractions containing the PEG20-clone-PEG20 are pooled andare further purified to removed mono-pegylated clone. This is achievedby cation exchange chromatography (SP; GE). A SP-HiTrap column ispre-equilibrated with Buffer A (20 mM NaH₂PO₄, 10 mM NaCl, pH 6.7), theSEC eluate is applied, the column is then washed with buffer A, and thena gradient from 0-10% buffer B (20 mM NaH₂PO₄, 1.0M NaCl, pH 6.7) over 5column volumes and held for an additional 5 column volumes is run.PEG20-clone-PEG20 is eluted from the SP-column by eluting with 50%Buffer B. Fractions are pooled by A280 and buffer exchanged into PBS byG25 Chromatography (GE).

BIAcore Analysis of the Soluble Fibronectin-Based Scaffold Proteins:

The binding kinetics of fibronectin-based scaffold proteins bindingproteins to the target is measured using Biacore 3000 or T100 biosensors(GE Healthcare, Piscataway, N.J.). Anti-human antibody (GE Healthcare,Piscataway, N.J.) is directly immobilized on Flow cells 1 and 2 (Fc1 andFc2) of Biasensor CM5 chip following the manufacturer's instructions.The kinetic analysis involves the capture of EGFR-Fc (R&D Systems,Minneapolis, Minn.) or the in-house generated truncated EGFR 525-Fc onanti-human IgG on Fc2 followed by injection of the clone in solution onFc1 and Fc2. The anti-human antibody surface is regenerated by 2successive injections of 3M MgCl₂. Sensorgrams are obtained at eachconcentration and are evaluated using the manufacturer's programBiaevaluation or Biacore T100 software, to determine the rate constantsk_(a) (k_(on)) and k_(d) (k_(off)). The dissociation constant, K_(D) iscalculated from the ratio of rate constants k_(off)/k_(on). Typically, aconcentration series (0 μM to 2 μM) of purified clone diluted in therunning buffer HBS-EP (10 mM Hepes 150 mM NaCl 3 mM EDTA 0.05%Surfactant P20) is evaluated for binding to anti-human IgG capturedhuman EGFR-Fc fusion protein.

For experiments determining binding to other related family member suchas HER2 or HER3, recombinant ectodomain-Fc fusions are captured usinganti-human IgG antibody as described above. Specific binding to eitherhuman EGFR, or other related family members such as HER2 or HER3 iscalculated by subtracting the binding observed to the blank referenceflow cell 1. VEGFR2-Fc, a non-related receptor, serves as an additionalnon-specific binding reference. EGFR binding clones are diluted to 10 μMin HBS-EP (10 mM Hepes 150 mM NaCl 3 mM EDTA 0.05% Surfactant P20) andare injected at 20 uL/min for 5 minutes over the flow cells at 25 C anddissociation is observed over 10 mins.

Differential Scanning Calorimetry:

Differential Scanning calorimetry (DSC) analysis of midscaled clones isperformed to determine thermal stability. A 1 mg/ml solution ofappropriate clone is scanned in a N-DSC II calorimeter (calorimetrySciences Corp) by ramping the temperature from 5° C. to 95° C. at a rateof 1 degree per minute under 3 atm pressure. The data is analyzed vs. acontrol run of the appropriate buffer using a best fit using OrginSoftware (OrginLab Corp).

Size-Exclusion Chromatography:

Size-exclusion chromatography (SEC) is performed using a TSKge1 SuperSW2000 column (TOSOH Biosciences, LLC), 4.6 mm×30 cm, on an Agilent 1100HPLC system with UV detection at A214 nm and A280 nm and withfluorescence detection (excitation=280 nm, emission=350 nm). A buffer of100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium chloride,pH 6.8 at a flow rate of 100 μL/min is employed. Samples (0.1 to 1 μgeach) at a concentration of approximately 100 μg/mL are injectedseparately. Gel filtration standards (Bio-Rad Laboratories, Hercules,Calif.) are used for molecular weight calibration.

SEC MALLS Analysis of Clones:

Size Exclusion Chromatography (SEC) is performed on a Waters Breeze HPLCsystem equipped with a Waters 2487 UV detector using a Superdex 200column (GE Healthcare) with a mobile phase of 100 mM Sodium Sulfate, 100mM Sodium Phosphate, 150 mM Sodium Chloride pH 6.8 applied at a flowrate of 0.6 ml/min. Samples are diluted to approximately 1.0 mg/ml withmobile phase and 50 μl is injected. Multi-Angle Laser Light Scattering(MALLS) analysis is performed using a miniDAWN Light Scattering detector(Wyatt Technology Corporation) and Optilab DSP DifferentialRefractometer (Wyatt Technology Corporation) plumbed in-line after theUV detector. Analysis of the light scattering data is performed usingAstra V version 5.1.9.1 software (Wyatt Technologies Corporation). Tocalculate the concentration of the clone by absorbance at 280 nm, atheoretical molar extinction coefficient based on amino acid sequence isused. For concentration determination by Refractive Index, an estimatedspecific refractive index increment (dn/dc) of 0.185 mL/g is used.

MALDI TOF Mass Spectrometry:

EGFR binding clones are analyzed by Matrix Assisted Laser DesorptionIonization Time of Flight (MALDI-TOF) mass spectrometery (FIG. 14) usinga Voyager DE PRO mass spectrometer (Applied Biosystems). Samples arediluted to approximately 1.0 mg/ml with 0.1% TFA. Approximately 12 μl ofsample is loaded onto a C4 ZipTip (Millipore Corporation) and is washedwith 0.1% Trifluoroacetic Acid (TFA) to remove salts and contaminants.Sample is eluted directly from the ZipTip onto the target plate using 2μl of Sinapinic Acid matrix (10 mg/ml in 70% Acetonitrile, 0.1% TFA).Standardization of the instrument is performed using two proteins ofknown mass: Cytochrome C (12361.96 Da) and Apomyoglobin (16952.27 Da)prepared to a final concentration of 5 μM in Sinapinic Acid and spottedonto the plate. Spectra are acquired with the following instrumentsettings: Accelerating Voltage 25000V, Grid Voltage 91%, Guide Wire0.1%, Extraction Delay Time 400 nsec, Laser Intensity 3824. Raw spectraare processed in Data Explorer v. 4.5 (Applied Biosystems) by applyingbaseline correction and the Gaussian Smooth algorithm with a filterwidth value of 9.

³H-Thymidine Cell Proliferation Assay

Cells were plated at 2,500 cells per well in 96-well microtiter plates.Compounds solubilized in PBS were added 24 hours later and the cultureswere incubated for an additional 72 hours. Cells were pulsed with 4uCi/mL [³H] thymidine (Amersham Pharmacia Biotech, Buckinghamshire,United Kingdom) for 3 hours, trypsinized, and harvested onto UniFilter-96, GF/B plates (Perkin-Elmer, Boston, Mass.). Incorporation intoDNA was measured by scintillation counting on a TopCount NXT (Packard,Meriden, Conn.). Results are expressed as an IC₅₀, which is the drugconcentration required to inhibit cell proliferation by 50% to that ofuntreated control cells. Data are averages of triplicate wells with SEsindicated.

Water Soluble Tetrazolium Salt Proliferation Assay

Cells were seeded in complete media without phenol red and treated withEGFR binding clones as described above. At the end of the treatmentperiod, 10 ul/well of the WST-8 reagent (Dojindo Molecular Technologies,Gaithersburg Md.) was added to each well and the plate was incubated forthree hours at 37 degrees in 5% CO₂. Accumulation of formazan dye wasquantified by reading absorbance at 450 nm on a SpectraMAX Plus(Molecular Devices, Sunnyvale, Calif.).

In Cell Western Assay

Cells were seeded into poly-D-lysine coated microtiter plates (BectonDickinson, Franklin Lakes, N.J.) at 24,000 cells/well for A431epidermoid carcinoma or FaDu head & neck carcinoma cells and allowed toadhere overnight. Cells were washed and then incubated for 24 hours inserum free media. Serial dilutions of clones were then applied to thecells and incubated for 4 hours prior to stimulation with 100 ng/ml EGFfor 10 minutes. Following stimulation, cells were fixed for 20 minutesin PBS containing 3.7% formaldehyde and then permeabilized in PBScontaining 0.1% triton-X-100 for 15 minutes. Cells were blocked for onehour in Odyssey blocker and incubated with antibodies to detect eitherEGFR phosphorylated on tyrosine 1068 (Cell Signaling, Beverly, Mass.)and actin (Sigma, St. Louis, Mo.) or ERK (MAP kinase phosphorylated ontyrosine 202/threonine 204 and total ERK (Santa Cruz Biotechnology,Santa Cruz, Calif.). After washing three times in PBS containing 0.1%tween-20, secondary antibodies were added (Invitrogen, Carlsbad, Calif.or Rockland, Gilbertsville, Pa.). Cells were washed three times in PBScontaining 0.1% tween-20 and imaged on a Li-Cor Odyssey Infrared ImagingSystem (Li-Cor Biosciences, Lincoln, Nebr.). Each clone was assayed intriplicate and IC₅₀ values were calculated from linear regressionanalysis of percent inhibition of maximum signal minus background.

Epitope Mapping by Blocking EGFR Antibodies

A431 cells were seeded into poly-D-lysine coated microtiter plates at24,000 cells/well and allowed to adhere overnight. The next day eachwell was washed once with cold PBS and cells were preincubated withvarious concentrations of clones for 1 hour at 4° C. Unbound protein waswashed away with cold PBS and bound protein was crosslinked to thereceptor by treating with 1 mM BS3 in PBS pH=8.0 at 4° C. for 1 hour.The contents of the plate were dumped and the crosslinking reaction wasquenched by adding 50 ul per well of 50 mM Tris-HCl pH=7.5 andincubating for 15 minutes at room temp. The plate was washed once inPBS+0.1% tween-20 and fixed for 20 minutes in PBS+3.7% formaldehyde. Theplate was blocked in Odyssey blocker for 1 hour at room temp. Blockingsolution was removed and various primary antibodies diluted 1:100 or1:50 in Odyssey blocker were added to the plate and incubated for 1 hourat room temp. The plate was washed three times and secondary antibodiesdiluted 1:800 in Odyssey blocker+0.2% Tween-20 along with TOPRO3counterstain 1:3000 were added to the plate and incubated for 1 hour atroom temp. The plate was washed three times and imaged on a Li-CorOdyssey infrared imaging system at 160 uM resolution.

ELISA Assay for Determination of Total EGFR Phosphotyrosine Levels inDiFi Cells

Inhibition of EGF-stimulated EGFR phosphorylation was determined byplating 1×10⁵ DiFi cells in each well of a microtiter plate and allowingthem to adhere overnight. The next day, media was replaced with serumfree media and cells were starved for 16 hours. EGFR binding clones orcontrol antibodies were incubated with cells at 37° C. for 4 hours.Cells were then stimulated with 20 ng/ml of EGF for 10 minutes and celllysates were prepared in HNTG [50 mM Hepes, 150 mM NaCl, 0.5%triton-X-100, 8% glycerol, 2 mM Na₃PO₄, 1.5 mM MgCl₇, 1 mM EDTAcontaining the protease inhibitors AEBSF, aprotinin, leupeptin,bestatin, pepstatin-A and E64]. Lysates were transferred to a captureplate coated with a primary antibody specific for the humanextracellular domain of the EGF receptor (Cell Signaling, Beverly,Mass.). The detection antibody was replaced with a mouse monoclonalantiphosphotyrosine antibody conjugated to horseradish peroxidase (4G10,Upstate Biotechnology, Lake Placid, N.Y.). The chromogenic substrate,tetra-methylbenzidine, was used to measure the absorbance on aspectrophotometer at 450 nm. Samples were tested in triplicate and IC₅₀values were determined by subtracting background and calculating percentinhibition of total maximum signal in each assay.

1. A polypeptide comprising a fibronectin type III (Fn3) domain, whereinthe Fn3 domain (i) comprises a loop, AB; a loop, BC; a loop, CD; a loop,DE; a loop EF; and a loop FG; (ii) has at least one loop selected fromloop BC, DE, and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human Fn3 domain, and (iii)binds human epidermal growth factor receptor (EGFR) with adisassociation constant of less than 10⁻⁴M.
 2. The polypeptide of claim1, wherein the Fn3 domain binds human EGFR with a disassociationconstant of less than 10⁻⁶M.
 3. The polypeptide of claim 1, wherein loopBC and loop FG have an altered amino acid sequence relative to thesequence of the corresponding loop of the human Fn3 domain.
 4. Thepolypeptide of claim 1, wherein the Fn3 domain is a tenth fibronectintype III domain (¹⁰Fn3).
 5. The polypeptide of claim 4, wherein the¹⁰Fn3 domain comprises a polypeptide selected from: (i) a polypeptidecomprising the amino acid sequence of any of one of SEQ ID NOS: 207-231;and (ii) a polypeptide comprising the amino acid sequence at least 90%identical to any of one of SEQ ID NOS: 207-231.
 6. The polypeptide ofclaim 1, further comprising one or more pharmacokinetic (PK) moietiesselected from: a polyoxyalkylene moiety, a human serum albumin bindingprotein, sialic acid, human serum albumin, IgG, an IgG binding protein,transferrin, and an Fc fragment.
 7. The polypeptide of claim 6, whereinthe PK moiety is the polyoxyalkylene moiety and said polyoxyalkylenemoiety is polyethylene glycol.
 8. The polypeptide of claim 6, whereinthe PK moiety and the Fn3 domain are operably linked via at least onedisulfide bond, a peptide bond, a polypeptide, a polymeric sugar, or apolyethylene glycol moiety.
 9. The polypeptide of claim 8, wherein thePK moiety and the Fn3 domain are operably linked via a polypeptidecomprising the amino acid sequence of SEQ ID NOS: 232-235.
 10. Thepolypeptide of claim 1, further comprising a second domain selectedfrom: an antibody moiety; a derivative of lipocalin; a derivative oftetranectin; an avimer; a derivative of ankyrin; and a secondfibronectin type III (Fn3) domain, wherein the second domain binds to ahuman protein, and wherein the second Fn3 domain (i) comprises a loop,AB; a loop, BC; a loop, CD; a loop, DE; and a loop FG; (ii) has at leastone loop selected from loop BC, DE, and FG with a randomized amino acidsequence relative to the sequence of the corresponding loop of the humanFn3 domain, and (iii) binds a human protein that is not bound by thehuman Fn3 domain.
 11. The polypeptide of claim 10, wherein the seconddomain is a second Fn3 domain, and wherein the second Fn3 domain bindsthe human protein with a disassociation constant of less than 10⁻⁴M. 12.The polypeptide of claim 10, wherein the second domain is a second Fn3domain, and wherein the second Fn3 domain is a tenth fibronectin typeIII domain (¹⁰Fn3).
 13. The polypeptide of claim 10, wherein the humanprotein bound by the second domain is selected from IGF-IR, EGFR, orVEGFR2.
 14. The polypeptide of claim 12, wherein the second ¹⁰Fn3 domaincomprises a polypeptide selected from: (i) a polypeptide comprising theamino acid sequence of any of one of SEQ ID NOS: 2-231 and 236; and (ii)a polypeptide comprising the amino acid sequence at least 90% identicalto any of one of SEQ ID NOS: 2-231 and
 236. 15. The polypeptide of claim10, wherein the Fn3 domain and the second domain are operably linked viaat least one disulfide bond, a peptide bond, a polypeptide, a polymericsugar, or a polyethylene glycol moiety.
 16. The polypeptide of claim 10,wherein said polypeptide inhibits the binding of transforming growthfactor alpha (TGF-alpha) or epidermal growth factor (EGF) to EGFR anddoes not activate human EGFR at sub IC₅₀ concentrations in a cell-basedassay.
 17. The polypeptide of claim 10, wherein said polypeptidecompetes with an anti-EGFR antibody for binding to EGFR.
 18. Thepolypeptide of claim 10, wherein said polypeptide inhibits totalEGF-stimulated phosphotyrosine activation of EGFR with an IC₅₀ of lessthan 10 μM.
 19. The polypeptide of claim 10, wherein said polypeptideinhibits ERK phosphorylation with an IC₅₀ of less than 10 μM.
 20. Thepolypeptide of claim 10, wherein said polypeptide inhibits AKTphosphorylation with an IC₅₀ of less than 10 μM.
 21. The polypeptide ofclaim 10, wherein said polypeptide has been deimmunized to remove one ormore T-cell epitopes.
 22. The polypeptide of claim 1, wherein said Fn3domain is selected by the method comprising the steps of a) producing apopulation of candidate RNA molecules, each comprising a candidate tenthfibronectin type III (Fn3) domain sequence which differs from human Fn3domain coding sequence, said RNA molecules each comprising a translationinitiation sequence and a start codon operably linked to said candidateFn3 domain coding sequence and each being operably linked to a nucleicacid-puromycin linker at the 3′ end; b) in vitro translating saidcandidate Fn3 domain coding sequences to produce a population ofcandidate RNA-Fn3 fusions; c) contacting said population of candidateRNA-Fn3 fusions with EGFR; and d) selecting an RNA-Fn3 fusion, theprotein portion of which has a binding affinity or specificity for EGFRthat is altered relative to the binding affinity or specificity of saidhuman Fn3 for EGFR.
 23. A pharmaceutically acceptable compositioncomprising the polypeptide of claim 1, wherein the composition isessentially endotoxin free.